To provide a mathematical tool to describe the combi nation effect of two agents, we performed fixed ratio dilu tion experiments to create isobolograms using a method of Chou and Talalay. Cells were treated with the single selleck Crenolanib agents and fixed ratios of NVP BGT226 or NVP BEZ235 plus sunitinib or imatinib to assess for induction of apoptosis. This was used to create isobolograms. Combination of NVP BGT226 with sunitinib in MOLM14 cells resulted in an experiment point that falls to the left of the predicted line of additive effect when taking ED90 as the experimental end point. Similar results were achieved for NVP BGT226 combined with imatinib in K562 cells with an experiment point lying on or falling to the left of the predicted line of additive effect.

Calculation of combination indices revealed Inhibitors,Modulators,Libraries a CI close to 1 for ED50s in both cell lines and a CI 1 for ED90 indicating Inhibitors,Modulators,Libraries synergy. Due to the moderate proapoptotic effect of NVP BEZ235 when administered as single agent, calculation of isobolograms and resultant CIs were restricted to ED25 50 concentrations for NVP BEZ235 TKI combinations. Nevertheless, a strong Inhibitors,Modulators,Libraries synergistic effect was revealed for both combinations of NVP BEZ235 plus sunitinib in FLT3 ITD positive MOLM14 cells, or NVP BE235 plus imatinib in BCR ABL1 positive K562 cells with CIs well smaller than 1. Additionally, estimated ED90s are provided along with each figure as well. These findings indicate that a combination approach may override the G1G0 arrest observed for NVP BEZ235 monotherapy which is supported by increased cleavage of caspase 3 in the western immunoblot experiments when combined with TKI.

Leukemia driving tyrosine kinase mutations trigger consecutive AKT serine phosphorylation of codon 473 and threonine phosphorylation of codon 308 Inhibitors,Modulators,Libraries In order to minimize cell type specific off target effects to validate our findings for the mutant FLT3 ITD cell line MOLM14 and the BCR ABL1 positive cell line K562, we established an isogenic BaF3 cell line model transfected with AKT autoactivating FLT3 ITD or BCR ABL1 mutations. We further comparatively Inhibitors,Modulators,Libraries ex tended our studies to additional leukemia associated mutant TK. Immunoblotting for phospho AKT was performed after successful transfection and weaning of IL3 dependent growth and found that AKT activation increases following website after transfection of plasmid vectors encoding for a FLT3 ITD, FLT3 D835V, KIT D816Y or BCR ABL1 isoform. While cytokine starved parental BaF3 cells did only re veal moderate, if any, phosphorylation levels of AKT, IL3 stimulated or oncogene transfected BaF3 cells did globally activate AKT on codons Thr308 as well as Ser473.

This may eliminate Alix from nascent VLP and impeded its ability to function in HIV 1 release in PTAP deficient strains of HIV. On the other hands, Alix also interacts with the nucleocapsid domain of HIV 1 Gag in addition Ixazomib Proteasome inhibitor to binding the LYPXnL motif, there Inhibitors,Modulators,Libraries by linking Gag to components of ESCRT III. Therefore, further analysis is needed to fully understand the molecular link between Gag phosphorylation and virus release through the AlixLYPXnL pathway. We further explored the physiological significance of Vpr Inhibitors,Modulators,Libraries incorporation into virions. Our current results clearly demonstrate that the inhibition of aPKC mediated Vpr incorporation prominently reduces the viral infectivity in MDMs. These results together indicate that Gag phos phorylation by aPKC plays a crucial role in the HIV 1 infection of macrophages.

aPKC has been demonstrated to be involved in Inhibitors,Modulators,Libraries cell po larity and migration in a number of study models. During cell migration, aPKC localizes on the leading edge of the plasma membrane where HIV 1 Gag is also loca lized in infected cells. It has been reported in an earlier study that aPKC is located at an immunological synapse with potential importance in cell to cell viral transfer. It is thus plausible that aPKC may regulate the incorpor ation Inhibitors,Modulators,Libraries of Vpr into virions at the leading edges or the HIV 1 virological synapse in polarized cells. It would be interesting to investigate whether aPKC cooperates with other factors in polarized HIV 1 infected cells in an additional mechanism to its function in Gag phosphorylation.

In the earlier study by Folgueira et al, it was de monstrated that aPKC mediates the NF ��B transcrip tional activation required for HIV 1 infection Inhibitors,Modulators,Libraries in U937 cells. It is of particular interest that aPKC is a one of the key regulators of HIV 1 infection. Our present findings also provide evidence for the involvement of aPKC in HIV 1 replication by showing that it directly phospho rylates Gag on Ser487, and that this phosphorylation mediates Vpr incorporation into virions. The targeting of aPKC activity is therefore a potential option as a novel therapeutic intervention against HIV 1 infection in com bination with existing anti retroviral treatments. Conclusions We have identified aPKC as a host protein kinase that phosphorylates HIV 1 Gag at its Ser487 residue.

Com puter assisted structural modeling and subsequent bio chemical assays revealed that the phosphorylation of Gag Ser487 enhances the association of Gag with Vpr and promotes the resultant incorporation of PF01367338 Vpr into virions. These events facilitate viral infectivity in macrophages. Hence, aPKC inhibition is a potential new therapeutic approach against HIV 1 infection in human macrophages. Methods Viral DNA constructs and plasmids The HIV 1 reporter virus vectors pNL4 3Env Luc and pNL4 3EnvVpr Luc were provided by Akifumi Takaori Kondo. The HIV 1 recombinant molecular clone pHIV 189. 6 and pHIV 1NLAD8 were provided by Akio Adachi.

Additionally, TGF B1 treatment was discovered significantly promoted the proliferation of ASMCs. However, wortannin, PD98059 and B CD alone or combination with TGF B1 could inhibit the proliferation remarkably. No significant difference was observed between inhibitor groups and TGF B1 combining with inhibitor groups. Caveolae and caveolin 1 in ASMCs By using SEM, we Navitoclax Bcl-xL discovered that caveolae structures in ASMCs between control and asthmatic group showed sig nificant differences. More caveolae were observed in con trol group than in asthmatic group. Moreover, TGF B1 stimulation. Over time, activation of ERK path way reached to almost twofold of time zero at 20 min, and then the level lowered and sustained at 60 min. While, activation of Inhibitors,Modulators,Libraries AKT pathway reached its peak at 20 min and nearly maintained at the peak level at 60 min.

Mechanisms of TGF B1 induced reduction in caveolin 1 and the effect of altered caveolin 1 expression on PI3K AKT and ERK12 regulation Western blot analysis of ASMCs demonstrated a signifi cantly reduced expression of caveolin 1 with TGF B1 ex posure, and then caveolin 1 level was increased Inhibitors,Modulators,Libraries slightly after treated with either 10 uM PD98059 or 0. 4 uM wortmannin. This result indicated that TGF B1 inhibited caveolin 1 expression partly through PI3K and ERK12 pathways. In addition, the Inhibitors,Modulators,Libraries caveolae in cultured Inhibitors,Modulators,Libraries ASMCs were destroyed by B CD with the expression of activated p AKT and p ERK12 significantly increased, followed by decreased expression of caveolin 1. According to our results, de creased caveolin 1 expression was downstream of ERK and AKT activation.

Thus, inhibition of ERK and AKT would inhibit TGF B1 induced down regulation of Caveolin 1. The effect of RXM on AKT and ERK12 activation and caveolin 1 expression Inhibitors,Modulators,Libraries The effect of RXM on caveolin 1 expression in ASMCs was assessed by western blot. Compared with TGF B1 group, RXM significantly increased the expression of caveolin 1 protein. In addition, the ex western blot was used to analyze caveolin 1 which mainly expressed in ASMCs. Compared with control group, the expression of caveolin 1 was significantly decreased in asthmatic group. The time course of ERK and AKT activation in ASMCs stimulated with TGF B1 The effect of TGF B1 on activation and expression of ERK and AKT was measured and our results indicated that ASMCs had a low level of p ERK12 at the beginning of pressions of p AKT and p ERK12 were significantly stimu lated by TGF B1 in ASMCs, and remarkably down regulated by RXM In a word, RXM treatment inhibited TGF B1 induced activation of AKT and ERK12 and down regulation of caveolin 1.

Discussion Our present study indicates that RXM treatment inhibits TGF B1 induced activation of ERK and AKT and down regulation of Caveolin 1 in ASMCs, which is an interest ing novel finding selleck chemicals about the potential mechanisms of RXM protection from chronic inflammatory diseases, in cluding bronchial asthma seen in clinical studies.

It is important to define the contribu tion of each pathway both to fully understand ruxolitinib structure cell survival signaling and to validate individual pathways as thera peutic targets. Activation of the Raf/MEK/ERK pathway has been often associated with the promotion of cell proliferation but also represents, in addition to the PI3K/Akt path way, an important survival signaling pathway in many tumor cells. The Raf/MEK/ERK pathway promotes survival through the inhibition of the apoptotic cascade by controlling the expression or the activity of Bcl 2 family members. There is evidence that the ERK pathway activation increases the expression of prosurvi val Bcl 2 proteins, notably Mcl 1, by promoting de novo gene expression. The relative expression of Mcl 1 in tumor cells can be regulated at the transcrip tional level or through post translational modifications by ERK.

In addition Inhibitors,Modulators,Libraries to the ERK signaling, the PI3K/ Akt pathway has been found to be critical for Mcl 1 ex pression. The importance of Mcl 1 in mediating tumor necrosis factor related apoptosis inducing ligand resistance Inhibitors,Modulators,Libraries has been well documented in differ ent cell types. Overexpression of Mcl 1 can attenu ate apoptosis induced by TRAIL. Conversely, downregulation of Mcl 1 by siRNA enhances TRAIL mediated cell death. TRAIL belongs to the TNF family of cytokines and has emerged as a promising anticancer agent, because of its ability to Inhibitors,Modulators,Libraries selectively induce apoptosis in a broad host of tumor cells. TRAIL binding to its receptors initiates the extrinsic path way, resulting in recruitment of the adapter protein Fas associated Inhibitors,Modulators,Libraries death domain and procaspase 8 in the death inducing signaling complex.

In some cells, the apoptotic signal from active caspase 8 is sufficient to activate downstream effector caspases and induce apoptosis. However, in other cell types, such as OC cells, the apoptotic signal must be further amplified by engaging the intrinsic Inhibitors,Modulators,Libraries pathway. In this context, caspase 8 cleaves Bid to generate Carfilzomib msds an active tBid, which in turn activates proapoptotic Bax or Bak proteins, and induces mito chondrial outer membrane permeabilization. The mitochondria then releases proapoptotic factors that promote effector caspase activation. Overexpression of antiapoptotic Bcl 2 family members, including Bcl 2, Bcl XL and Mcl 1 is associated with TRAIL resistance in type II cells, because of their ability to prevent tBid induced MOMP. In this study, we demonstrate that transcriptional upregulation of Mcl 1 by OC ascites is mediated by an ERK dependent activation of the transcription factor Elk 1. Moreover, we demonstrate that upregulation of Mcl 1 has a significant role in ascites mediated attenu ation of TRAIL induced apoptosis.

Protein concentration was deter mined by BCA assay. Protein selleck chemical Axitinib was precleared with Protein A Sepharose CL 4B on a Inhibitors,Modulators,Libraries rotator at 4 C for 1. 5 h. Pre cleared supernatant was collected and immunopre cipitated overnight with anti HDAC3 or anti HDAC6 rab bit polyclonal antibody. Protein A Sepharose beads were collected and washed before immunoblotting with anti HDAC3, anti SMRT, anti phosphoSMRT, anti Pin1, anti 14 3 3, and anti casein kinase IIa antibodies. The superna tant depleted of HDAC3 and/or HDAC6 was collected and kept frozen at 80 C until used for HDAC activity assays. In some experiments, HDAC3 pulls downs were followed by immunoblotting for p 14 3 3 and p 14 3 3, both at 1 250 dilution. Overexpression and knock down experiments HDAC3 and HDAC6, as transfection ready DNA in pCMV6 XL4 vector, and Pin1 siRNA and control siRNA were from Origene.

Cells were transfected using Lipofectamine 2000 at a ratio of 1 3 1 4 in reduced serum med ium according to the manufacturers protocol. Inhibitors,Modulators,Libraries SFN treatment Inhibitors,Modulators,Libraries started after 24 h of transfection. Immunoblotting was carried out with whole cell lysates prepared using lysis buffer. Statistics The results of each experiment shown are representative of at least three independent assays. Where indicated, results were expressed as mean standard error, and differences between the groups were deter mined using Students t test. For multiple comparisons, ANOVA followed by the Dunnetts test was performed using GraphPad Prism. A p value 0. 05 was considered as statistically significant, and indicated as such with an asterisk in the corresponding figure.

Background Lung cancer is a worldwide epidemic. In 2009, nearly 160,000 people died from lung cancer in the U. S. alone. The five year survival rate slightly increased from 13% to 15% over the last 25 years, mainly due to limited early cancer detection and minor improvements in ther apy. Non small cell lung cancer is the most common form of the disease, and adenocarcinoma of the distal Inhibitors,Modulators,Libraries lung the most frequently diagnosed subtype. Persistent Inhibitors,Modulators,Libraries lung inflammation due to cigar ette smoke and related pulmonary comorbidities such as chronic obstructive pulmonary disease increases the life time risk of developing lung cancer, which can be partially alleviated by long term anti inflammatory drug therapy. Therefore, delineating the causal relation ship between inflammation and lung carcinogenesis may lead to earlier diagnosis and more effective treatment.

To understand how chronic lung inflammation pro motes the growth of lung cancer, it is important to examine communication between pulmonary epithelial cells and inflammatory effector cells such as alveolar macrophages. Macrophages are the most abundant type of immune cell in a healthy lung, screening libraries and alveolar macrophage numbers increase dramatically as chronic diseases like NSCLC progress.

Treatment of Fuji cells with U0126 led to the marked inhibition of P5 activity. No cell toxicity was observed concerning mor phology and selleck chemical growth of both cell lines under the conditions of this experiment. Consis tently, U0126 also markedly decreased the expression of CD133 protein in Fuji cells, indicating that the MEK/ERK signaling is implicated in P5 mediated CD133 expression. In contrast, Inhibitors,Modulators,Libraries the same concentration of U0126 could not affect P5 activity and expression of CD133 in Caco 2 cells, suggesting that another pathway could regulate Ets mediated P5 transcription in Caco 2 cells. However, it remains possible that the ERK path way might simultaneously regulate other promoters. In fact, ERK inhibition decreased the expression of exon 1B and 1D and 1E containing CD133 mRNA, but increase exon 1C containing one.

These data indicate cell type specific regula tion of CD133 gene expression by the ERK pathway. Inhibition of MEK/ERK pathway abolishes side population in Caco 2 cells To assure the relevance of the Inhibitors,Modulators,Libraries ERK pathway to stem like characteristics, the effect of U0126 on the amount of SP was assessed by flow cytometric analysis. The SP frac tion in tumor cells has been known to define the popu lation containing stem like cells, which highly express ATP binding cassette transporters to efflux both Hoechst dye and chemotherapeutic agents, and to have a high capacity to form tumor xenografts in mice. In our experiments, the side population represented approximately 2% in the Caco 2 cell line. Treatment with verapamil, an inhibitor of the ABC transporters, completely ablated this popula tion.

In contrast, no distinct SP was visible in the Fuji cell line. Treatment of Caco 2 with U0126 dramatically reduced the SP frequency to 0. 15%. This result emphasized our con clusion that ERK is a key molecule in the signal trans duction to maintain stem like features in tumor Inhibitors,Modulators,Libraries cells. Ets2 increases CD133 Inhibitors,Modulators,Libraries mRNA levels in human astrocytes, but cannot confer tumorigenicity To examine whether Ets factor could increase CD133 expression and confer tumorigenicity in normal cells, we established the immortalized human astrocytes overex pressing Ets2. The increase of CD133 mRNA expression was detected in NHA/TSE2 compar ing to NHA/TS cells and its effect was strongly Inhibitors,Modulators,Libraries enhanced by the treatment with demethylating agent 5 Aza dC and histone deacetyltransferase inhibitor TSA.

5Aza dC/TSA treatment has been shown to open the chromosomal region to increase accessibility for transcription factor complexes different to assemble at the promoter and drive gene transcription. However, the elevated protein level of CD133 could not be detected by FACS analysis. Instead, approximately 0. 04% of side population diminished by verapamil was observed in the NHA/TSE2 cells, whereas substantial SP was not visible in NHA/TS cells.

After cleavage and removal of the DEVD peptides full read by caspase 3/7 activity, the fluorescence in each well was quantitated at an excitation wavelength of 485 20 nM and an emission wavelength of 535 25 nM and after correction based on blank control or the homogeneous caspase 3/7 reagent. Final fluorescent intensity was depicted as endpoint rela tive fluorescent Inhibitors,Modulators,Libraries unit, RFLU. Using similar experimental conditions Inhibitors,Modulators,Libraries but as an inde pendent study, the effect of LY294002 was also evaluated on growth and caspase 3/7 activity of cells treated with saposin C etoposide. After initial studies to find the optimal concentration, we used a non toxic tolerable dosage of 1. 5M for LY294002. In addition, we chose the most effective concen tration of etoposide and saposin C for this study.

Terminal deoxynucleotide transferase mediated nick end labeling Inhibitors,Modulators,Libraries Cells were cultured in multiwell chamber slides and treated with etoposide in the presence or absence of saposin C at 0. 1, 1, or 10 nM as indicated above. In situ determination of apoptosis by Terminal dUTP nick end labeling was performed using an ApopTag Per oxidase Inhibitors,Modulators,Libraries In Situ kit as recommended by the manufacturer. The ApopTag Kit detects single and double stranded DNA breaks asso ciated with apoptosis. Drug induced DNA damage is not identified by the TUNEL assay unless it is coupled with the apoptotic response. Briefly, at the end of the incubation period, cells were fixed in 1% paraformaldehyde in PBS, pH 7. 4 for 10 min at room temperature, washed with PBS twice, and permeabilized in pre cooled ethanol acetic acid for 5 min at 20 C.

After washing twice in PBS, 5 min each time, endogenous peroxidase activity in the cells was quenched in 3% H2O2 in PBS for 5 min at room temperature, incubated with terminal deoxynucleotidyl transferase and then with peroxidase con jugated Inhibitors,Modulators,Libraries anti digoxigenin antibody. Nuclear staining of the apoptotic cells was detected by 3,3 diaminobenzidine tetrahydrochloride dihydrate substrate, as recommended by the manufacturer. Cells were then counterstained in 0. 5% methyl green and slides were mounted under a glass coverslip in permount mounting medium. For control staining, the enzyme incubation step was deleted. Microscopic examination of cells was carried out using a phase contrast microscope. Cells were counted by choosing ten random fields and the molarity calculator percentages of apop totic cells were determined. Apoptosis was indicated by the presence of apoptotic bodies, exhibiting brightly labeled punctuated nuclei. Statistical analyses For cell survival and other quantitative data, a one way analysis of variance was employed to evaluate the influence of one variable on multiple independent groups. Bonferronis corrections were also applied when ever a significant group effect was observed.

For statistical analysis, SPSS Statistics Ver sion 18 was used. P values smaller cancers showed a low HDAC1 Tofacitinib FDA expression. animal study HDAC2 expression was correlated significantly Inhibitors,Modulators,Libraries with histological grade 43. 6% Inhibitors,Modulators,Libraries of the grade Inhibitors,Modulators,Libraries 3 tumors exhibited a high expression vs. 22. toward 8% and 10% for grade 2 and grade 1 tumors, respectively. In contrast, 56. 7% of the grade 1 tumors showed a low expression. Additionally, a high HDAC2 expression was significantly associated with a negative hormone receptor status and an overexpression of HER2 as well as the presence of nodal metastasis. A high HDAC3 expression was observed in less differ entiated tumors and tumors with negative hormone receptor status .

The remaining clinicopathological parameters revealed Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries no significant correlations.

The correlations of all three iso enzymes are shown in Tables 3, 4 and Inhibitors,Modulators,Libraries 5.

HDAC2 and HDAC3 show a strong positive correl Inhibitors,Modulators,Libraries ation. Correlation of Inhibitors,Modulators,Libraries HDAC isoforms with survival The known prognostic factors including nodal status, histopathological grading and pT status achieved statistical significance in this cohort. In contrast, none of the HDAC isoforms reached significant Inhibitors,Modulators,Libraries prognostic relevance in our study using Kaplan Meyer survival analysis. Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries Additionally, a co expression of HDAC2 and HDAC3 did also not reach significant prognostic relevance. Discussion Our study demonstrates a differential expression of HDAC1, HDAC2 and HDAC3 using immunohistochem istry in breast cancer.

Inhibitors,Modulators,Libraries Expression of all three isoforms re vealed significant correlations with clinicopathological parameters.

Expression of HDAC2 and HDAC3 was sig nificantly higher in less differentiated Inhibitors,Modulators,Libraries tumors as well as in tumors Inhibitors,Modulators,Libraries with negative hormone receptor status. Addition ally, tumors with HER2 overexpression and positive lymph node metastasis showed a significant higher expression of HDAC2. In contrast, a high expression of the HDAC1 was found in hormone Inhibitors,Modulators,Libraries receptor positive tumors. To our knowledge, this is the first time that the class 1 isoforms HDAC1, 2 and ?3 were analyzed together in the same breast cancer cohort. Krusche et al. did an immunhistochemical ana lysis of the expression of HDAC1 and HDAC3 in 200 breast cancer samples.

Similar to our findings, sellekchem they found a significant correlation between positive HDAC1 expression and positive hormone receptor expression.

In contrast to our results, they additionally described a cor relation of HDAC3 with a positive hormone receptor ex pression.

They found no significant results concerning the correlation of HDAC and grading. M��ller et al. Similarly with our findings, Zhang et al. 17-AAG solubility showed simi lar results concerning HDAC1, with an increased HDAC1 mRNA expression in hormone receptor positive tumors. Most interestingly, we could find a significantly selleck chemicals higher expression of HDAC2 and ?3 in more aggressive tumor types.

Bay11 7082 significantly attenuated the increased transcriptional activ ity of NF ��B driven sellekchem luciferase reporter in these two cell lines, thus confirmed the efficiency of Bay11 7082 as an NF ��B inhibitor. Notably, the increased tran scriptional activity of the Mcl 1 promoter observed in Eca109 cells remained unchanged by the above three strategies. Taken together, these results pro vide consistent evidence that the involvement of NF ��B pathway in the Mcl 1 promoter transcriptional activity in various human ESCC cells. NF ��B signaling pathway contributes to Mcl 1 expression in various human esophageal squamous cell carcinoma Inhibitors,Modulators,Libraries cell lines We further confirm whether NF ��B is involved in Mcl Inhibitors,Modulators,Libraries 1 expression in human ESCC cells. Bay11 7082 was firstly used to investigate the effect of NF ��B activation on Mcl 1 induction.

Treatment of TE 1 cells with the in hibitor resulted in a dose dependent attenuation of Mcl 1 induction. Similar results were obtained Inhibitors,Modulators,Libraries from KYSE150 cells treated with various concentrations Mcl 1 ��Bwt generated higher luciferase activity than that of the pGL2 Basic construct, indicated that high transcrip tional activity of human Mcl 1 promoter in three Mcl 1 expressing ESCC cell lines tested. However, with a pro moter construct mutated at the ��B site, the loss of Mcl 1 promoter activity was observed in TE 1 and KYSE150 cells. Dominant negative mutants of I��B, a truncant mutant with a deletion of 71 amino acids at the N terminus of I��B, can competitively inhibit the activation of NF ��B was used to block NF ��B activation Inhibitors,Modulators,Libraries as described previously.

Expression of DNMI��B significantly inhibited the Mcl 1 promoter ac tivity in TE 1 and KYSE150 cells. Further more, compared with their respective DMSO control, treatment with 20 uM Bay11 7082, a specific NF ��B in hibitor, resulted in the Mcl 1 promoter activity drastically curtailed in both TE 1 and KYSE150 cells. The activity of the Inhibitors,Modulators,Libraries Mcl 1 promoter with mutated NF ��B site was essen tially unaffected by inhibitor treatment. NF ��B transcriptional activities in both TE 1 and KYSE150 cell lines have also been estimated by using an NF ��B of Bay11 7082. DNMI��B was further used to test the role of NF ��B pathway in regulating Mcl 1 expression. As verified by Western blotting analysis, ex pression of DNMI��B in TE 1 or KYSE150 cells led to a significant decrease of Mcl 1 induction compared with the vector control.

The results suggested GW572016 that NF ��B pathway is involved in Mcl 1 ex pression in TE 1 and KYSE150 cells. Binding of transcription factor NF ��B family members to human Mcl 1 promoter To ascertain whether NF ��B transcription factor can bind the NF ��B site in human Mcl 1 promoter, EMSA was performed with an oligonucleotide probe containing the putative NF ��B binding sequence derived from hu man Mcl 1 promoter.