Journal: Oncotarget

Article Title: Yin Yang-1 increases apoptosis through Bax activation in pancreatic cancer cells

doi: 10.18632/oncotarget.8654

Figure Lengend Snippet: A. DNA fragmentation was measured by Cell Death ELISA. Results are representative of three independent experiments and are presented as the mean ± SD (bars). * p < 0.05; # p < 0.01. B. Changes in nuclear morphology were measured by Hoechst 33258 staining. Arrowheads indicate apoptotic cells characterized by morphological changes such as chromatin condensation and nuclear fragmentation. Results are representative of three independent experiments. C. Percentages of apoptotic cells in Annexin V-FITC/PI staining assay were detected by flow cytometry. Results are representative of three independent experiments and are presented as the mean ± SD (bars). * p < 0.05; # p < 0.01. D. Volumes of tumors grown from BXPC-3-Vector and BXPC-3-YY1 cells bilaterally injected into the flank region of the mice (1.5×10 6 cells/100 μl per flank). Data are presented as means ± SD of tumors for each group. * p <0.05. E. Apoptosis in mouse xenograft tumor tissues was detected by in situ TUNEL assay.

Article Snippet: Thermal cycling conditions consisted of an initial denaturation step at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. TaqMan Gene Expression Assay Mixes were used to detect YY1 and Bax (product numbers Hs00231533_m1 and Hs00180269_m1, respectively), while human GAPDH (product number Hs02758991_g1) was measured to calibrate the original mRNA concentration.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Plasmid Preparation, Injection, In Situ, TUNEL Assay

Journal: Oncotarget

Article Title: Yin Yang-1 increases apoptosis through Bax activation in pancreatic cancer cells

doi: 10.18632/oncotarget.8654

Figure Lengend Snippet: A. Cleavage of caspases-3 and -7 and PARP in YY1 overexpressing or knockdown BXPC-3 cells was measured by Western blot. Results are representative of three independent experiments. B. Cytochrome c localization to mitochondrial membrane and cytosolic fractions was measured by Western blot. Mitochondrial marker Cox-IV (cytochrome oxidase IV) was used as a loading control, and each blot was quantified by densitometry (right panel) to assess the translocation of proteins. Results are representative of three independent experiments.

Article Snippet: Thermal cycling conditions consisted of an initial denaturation step at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. TaqMan Gene Expression Assay Mixes were used to detect YY1 and Bax (product numbers Hs00231533_m1 and Hs00180269_m1, respectively), while human GAPDH (product number Hs02758991_g1) was measured to calibrate the original mRNA concentration.

Techniques: Knockdown, Western Blot, Membrane, Marker, Control, Translocation Assay

Journal: Oncotarget

Article Title: Yin Yang-1 increases apoptosis through Bax activation in pancreatic cancer cells

doi: 10.18632/oncotarget.8654

Figure Lengend Snippet: A. Bax expression in YY1 overexpressing or knockdown BXPC-3 and PANC-1 cells was measured by quantitative RT-PCR and Western blot. Results are representative of three independent experiments and are presented as the mean ± SD (bars). # p < 0.01. B. Bax expression in mitochondrial membrane and cytosolic fractions of BXPC-3 cells was measured by Western blot. Each blot was measured by densitometry (right panel) to assess the translocation of proteins. Results are representative of three independent experiments. C. YY1 overexpressing BXPC-3 cells were transfected with Bax siRNA for 48 h, after which the extent of apoptosis (left panel) and Bax protein expression levels (right panel) were measured. Results are representative of three independent experiments and are presented as the mean ± SD (bars). # p < 0.01.

Article Snippet: Thermal cycling conditions consisted of an initial denaturation step at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. TaqMan Gene Expression Assay Mixes were used to detect YY1 and Bax (product numbers Hs00231533_m1 and Hs00180269_m1, respectively), while human GAPDH (product number Hs02758991_g1) was measured to calibrate the original mRNA concentration.

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Western Blot, Membrane, Translocation Assay, Transfection

Journal: Oncotarget

Article Title: Yin Yang-1 increases apoptosis through Bax activation in pancreatic cancer cells

doi: 10.18632/oncotarget.8654

Figure Lengend Snippet: A. A scatter diagram for correlation between YY1 and BAX mRNA expression in 50 pancreatic cancer tissues. B. ROC curve for BAX expression and cut-off value selection for high and low level BAX expression. C. Kaplan-Meier survival curves for 50 patients with pancreatic cancer according to their BAX mRNA expression status. The p value was calculated by the Log-rank test.

Article Snippet: Thermal cycling conditions consisted of an initial denaturation step at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. TaqMan Gene Expression Assay Mixes were used to detect YY1 and Bax (product numbers Hs00231533_m1 and Hs00180269_m1, respectively), while human GAPDH (product number Hs02758991_g1) was measured to calibrate the original mRNA concentration.

Techniques: Expressing, Selection

Journal: Oncotarget

Article Title: Yin Yang-1 increases apoptosis through Bax activation in pancreatic cancer cells

doi: 10.18632/oncotarget.8654

Figure Lengend Snippet: A. Schematic diagram of the luciferase reporter construct containing the human Bax promoter (pBax) and the mutant construct (pBax-YY1-M) containing the Bax promoter in which the presumed YY1 binding site was mutated. B. Luciferase activity of Bax promoter in YY1 overexpressing BXPC-3 cells was increased compared with control cells. Results are representative of three independent experiments and are presented as mean ± SD (bars). # p <0.01. C. Luciferase activity of the Bax promoter was decreased when the presumed YY1 binding site (nucleotides -1022 to -1014) was mutated. Results are representative of three independent experiments and are presented as the mean ± SD (bars). # p < 0.01.

Article Snippet: Thermal cycling conditions consisted of an initial denaturation step at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. TaqMan Gene Expression Assay Mixes were used to detect YY1 and Bax (product numbers Hs00231533_m1 and Hs00180269_m1, respectively), while human GAPDH (product number Hs02758991_g1) was measured to calibrate the original mRNA concentration.

Techniques: Luciferase, Construct, Mutagenesis, Binding Assay, Activity Assay, Control

Journal: Oncotarget

Article Title: Yin Yang-1 increases apoptosis through Bax activation in pancreatic cancer cells

doi: 10.18632/oncotarget.8654

Figure Lengend Snippet: A. EMSA and supershift assay of YY1 binding to Bax promoter. The wild-type probe was incubated without (lane 1) or with (lane 2) BXPC-3-YY1 cell nuclear proteins in the absence or presence of unlabeled probe (lanes 3-6). Lanes 3 and 4 contain the wild-type probe, and lanes 5 and 6 contain the mutant probe, each at 50- and 100-fold molar excess. A supershift assay was performed using an anti-YY1 antibody (lane 7). B. ChIP assay of YY1 binding to Bax promoter. Lane 1, DNA marker; lane 2, input DNA; lane 3, DNA from BXPC-3-YY1 cells immunoprecipitated with anti-YY1 antibody; lane 4, DNA from BXPC-3-YY1 cells immunoprecipitated with normal rabbit IgG.

Article Snippet: Thermal cycling conditions consisted of an initial denaturation step at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. TaqMan Gene Expression Assay Mixes were used to detect YY1 and Bax (product numbers Hs00231533_m1 and Hs00180269_m1, respectively), while human GAPDH (product number Hs02758991_g1) was measured to calibrate the original mRNA concentration.

Techniques: Binding Assay, Incubation, Mutagenesis, Marker, Immunoprecipitation

Journal: Nature genetics

Article Title: Mutations in the evolutionarily highly conserved KEOPS complex genes cause nephrotic syndrome with microcephaly

doi: 10.1038/ng.3933

Figure Lengend Snippet: Primary Antibodies (Validation: Supplementary Fig. 24–25 )

Article Snippet: Clones reflecting the mutations identified in individuals with GAMOS were introduced in the cDNA constructed using the Quick change II XL site-directed mutagenesis kit (Agilent Technologies) following the manufacturer’s instructions. table ft1 table-wrap mode="anchored" t5 Target protein Company Catalogue # Species Dilution (IF) Dilution (WB) LAGE3 LS Bioscience LS-C201798 Rabbit 1:200 1:1,000 OSGEP Novus biologicals NBP2-00823 Mouse 1:400 1:1,000 TP53RK Abgent AP17010b-ev Rabbit 1:200 1:1,000 TP53RK Santa Cruz Biotechnology sc-85846 Goat 1:100 1:1,000 TPRKB Abcam ab128112 Mouse 1:200 n/a TPRKB Abcam ab96261 Rabbit n/a 1:1,000 ARP2 Abcam ab128934 Rabbit 1:200 1;1,000 Phospho-Histone H2A.X Cell Signaling (#9718) Rabbit 1:200 1;1,000 Histone H2A.X Cell Signaling (#2595) Rabbit n/a 1;1,000 PARP1 Sigma Aldrich HPA045168 Rabbit 1:200 1;1,000 p21 Waf1/Cip1 Cell Signaling (#2947) Rabbit n/a 1;1,000 BAX Cell Signaling (#5023) Rabbit n/a 1;1,000 Cleaved Caspase-3 (Asp175) Cell Signaling (#9661) Rabbit n/a 1;1,000 Synaptopodin American Research Products #03-65194 Mouse 1:200 n/a WT1 Santa Cruz Biotechnology sc-192 Rabbit 1:100 n/a Open in a separate window Primary Antibodies (Validation: Supplementary Fig. 24–25 ) Immunofluorescence and confocal microscopy in cell lines and tissue sections Immunostaining was performed as described previously in immortalized human podocytes cell lines.

Techniques:

Journal: Genes & Development

Article Title: The retinoblastoma protein induces apoptosis directly at the mitochondria

doi: 10.1101/gad.211326.112

Figure Lengend Snippet: pRB promotes mitochondrial apoptosis in a BAX-dependent manner. (A) pRB expression was induced for 24 h in stable pools of wild-type, Bak−/−, Bax−/−, and Bax−/−;Bak−/− immortalized MEFs by doxycycline addition and confirmed by Western blotting using antibodies against pRB, BAK, BAX, and tubulin. (B) Wild-type, Bak−/−, Bax−/−, and Bax−/−;Bak−/− immortalized MEFs with or without 24 h of pRB expression were left untreated or treated with TNFα and CHX for 10 h and analyzed for apoptosis by AnnexinV staining. Induction of pRB in wild-type and Bak−/−, but not Bax−/− or Bax−/−;Bak−/−, MEFs sensitized to TNFα/CHX-induced apoptosis. Each MEF variant was independently generated twice. Graph bars represent the average of three representative, independent experiments (±SD).

Article Snippet: The following antibodies were used in 2.5% nonfat milk: human pRB (Cell Signaling, 9309), rodent pRB (Becton Dickinson, 554136), phospho-pRB (Cell Signaling, 2181, 9301, 9307, and 9308), procaspase 7 (Cell Signaling, 9492), cleaved caspase 7 (Cell Signaling, 9491), procaspase 3 (Cell Signaling, 9662), cleaved caspase 3 (Cell Signaling, 9661), actin (Santa Cruz Biotechnology, SC1616 HRP), tubulin (Sigma, T9026), BAX (Cell Signaling, 2772; Santa Cruz Biotechnology, sc-493), BAK (Cell Signaling, 3814), cyclin A (Santa Cruz Biotechnology, sc594), HDAC1 (Upstate Biotechnology, 05-614), PCNA (Abcam, ab29), Lamin A/C (Cell Signaling, 2032), COXIV (Cell Signaling, 4850), Histone H3 (Santa Cruz Biotechnology, sc8654), ATPB (Abcam, ab5432), SirT3 (Cell Signaling, 5490), cytochrome c (Becton Dickinson, 556433), p16 (Santa Cruz Biotechnology, sc74401), HA.11 (Covance), and GAPDH (Ambion, 4300).

Techniques: Expressing, Western Blot, Staining, Variant Assay, Generated

Journal: Genes & Development

Article Title: The retinoblastoma protein induces apoptosis directly at the mitochondria

doi: 10.1101/gad.211326.112

Figure Lengend Snippet: Mitochondria targeted pRB is deficient for pRB's nuclear function but induces apoptosis in response to various apoptotic stimuli. (A) Schematic of mitoRBΔNLS construct. pRB was targeted to mitochondria by fusion to the mitochondrial leader peptide of ornithine transcarbamylase and mutation of the NLS. (B) Stable variants of RAT16 cells allowing for inducible expression of mitoRBΔNLS, wild-type pRB, mitoREL, and wild-type REL were generated, and cellular localization following 24 h of induction was assessed by immunofluorescence using antibodies against pRB and REL. Mitochondria were visualized using MitoTracker. mitoRBΔNLS, and mitoREL localized to mitochondria. (C) Western blotting showing induced expression levels of mitoRBΔNLS and wild-type pRB at mitochondria and total lysate. mitoRBΔNLS expressed at lower levels than wild-type pRB even when considering the mitochondrial fraction. (D) pRB, but not mitoRBΔNLS, repressed E2F target genes cdc2, mcm3, mcm5, mcm6, PCNA, and cycA2 as measured by RT-qPCR and normalized to ubiquitin. Average of two independent experiments (±SD). (E) Induced expression of mitoRBΔNLS and wild-type pRB, but not mitoREL or REL, sensitized to apoptosis induced by 24 h of treatment with TNFα and CHX. (B–E) Each RAT16 variant was independently generated three times. Graph bars represent the average of three representative, independent experiments (±SD). (F) mitoRBΔNLS expression was induced by doxycycline addition for 24 h in stable variants of wild-type, Bak−/−, Bax−/−, and Bax−/−;Bak−/− immortalized MEFs and confirmed by Western blotting using antibodies against pRB, BAK, BAX, and tubulin. (G) Induction of mitoRBΔNLS in wild-type and Bak−/−, but not Bax−/− or Bax−/−;Bak−/−, MEFs sensitized to 10 h of treatment with TNFα and CHX. (H) Wild-type and Bax−/−;Bak−/− MEFs with and without induced expression of mitoRBΔNLS were treated with STS (1 μM) for 6 h or etoposide (25 μM) for 12 h. Induction of mitoRBΔNLS in wild-type, but not Bax−/−;Bak−/−, MEFs enhanced apoptosis in response to STS and etoposide. (F–H) Each MEF variant was independently generated twice. Graph bars represent the average of three representative, independent experiments (±SD).

Article Snippet: The following antibodies were used in 2.5% nonfat milk: human pRB (Cell Signaling, 9309), rodent pRB (Becton Dickinson, 554136), phospho-pRB (Cell Signaling, 2181, 9301, 9307, and 9308), procaspase 7 (Cell Signaling, 9492), cleaved caspase 7 (Cell Signaling, 9491), procaspase 3 (Cell Signaling, 9662), cleaved caspase 3 (Cell Signaling, 9661), actin (Santa Cruz Biotechnology, SC1616 HRP), tubulin (Sigma, T9026), BAX (Cell Signaling, 2772; Santa Cruz Biotechnology, sc-493), BAK (Cell Signaling, 3814), cyclin A (Santa Cruz Biotechnology, sc594), HDAC1 (Upstate Biotechnology, 05-614), PCNA (Abcam, ab29), Lamin A/C (Cell Signaling, 2032), COXIV (Cell Signaling, 4850), Histone H3 (Santa Cruz Biotechnology, sc8654), ATPB (Abcam, ab5432), SirT3 (Cell Signaling, 5490), cytochrome c (Becton Dickinson, 556433), p16 (Santa Cruz Biotechnology, sc74401), HA.11 (Covance), and GAPDH (Ambion, 4300).

Techniques: Construct, Mutagenesis, Expressing, Generated, Immunofluorescence, Western Blot, Quantitative RT-PCR, Variant Assay

Journal: Oncotarget

Article Title: Drug-induced premature senescence model in human dental follicle stem cells

doi: 10.18632/oncotarget.14085

Figure Lengend Snippet: a . The key regulatory networks underlying the HU-induced aging model. Proteomic data were imported into the IPA and interacting pathways were constructed in protein expression. b . Western blotting analysis of lap2a, hp1a and wrn expression. c . Verification of SOD2 expression by RT-PCR with β-actin as an internal control. The data are presented as mean±S.E. from three independent experiments. d . Western blotting analysis of bal2 and bax expression. e . The schematic diagram outlines the key players and pathways that may mechanistically contribute to the phenotypes in our DFSC aging model.

Article Snippet: The primary antibodies used include rabbit anti-p16INK4A (Abclonal Technology, Wuhan, China), anti-p21 (Abclonal Technology, Wuhan, China), anti-p53 (Abcam), anti-ku70 (Abcam), anti-Xrcc2 (Abclonal Technology, Wuhan, China), anti-ligase4 (Abclonal Technology), anti-brca1 (Abclonal Technology),anti-Xrcc4 (Abclonal Technology) and mouse anti-β-tublin (Abclonal Technology, Wuhan, China), WRN (Abcam), Hp1a (Abcam), Lap2a (Abcam), BCL2 (Abcam), BAX (Abcam).

Techniques: Construct, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: Human VRK2 modulates apoptosis by interaction with Bcl-xL and regulation of BAX gene expression

doi: 10.1038/cddis.2013.40

Figure Lengend Snippet: Subcellular localization and interactions of VRK2A with apoptotic proteins. ( a ) Colocalization of endogenous VRK2 with transfected Bcl-xL detected by confocal microscopy in A549 cells. VRK2 was detected with a rabbit polyclonal antibody and Bcl-xL with a monoclonal antibody. ( b ) Colocalization of VRK2 with transfected Bax detected by confocal microscopy in A549 cells. Bax was detected with a monoclonal antibody. ( c ) Detection of interaction between VRK2 and Bcl-xL, Bcl-2 or Bax in pulldown assays. HeLa cells were transfected with plasmid pCEFL-GST-VRK2A that expresses GST-VRK2A and used in a pulldown assay in which the associated protein were detected in immunoblots using specific antibodies for GST that detects transfected GST and GST-VRK2, and specific antibodies for detection of endogenous Bcl-2, Bcl-xL or Bax. The ratio of GST-VRK2/actin ranges between 0.72–0.66 in the three bots. The ration of Bcl-xL/actin is 1.50, and the ratio Bax/actin is 1.40, respectively.( d ) Mapping the interaction between VRK2A and Bcl-xL. The region of interaction between VRK2A and Bcl-xL was performed by pulldown assays with different constructs of the VRK2A protein, which is summarized in a diagram to the right. VRK2B is a spliced variant that has 397 aminoa cids and is identical to the N-terminal region of VRK2A

Article Snippet: Rabbit polyclonal anti-Bax was from Abcam (Cambridge, UK).

Techniques: Transfection, Confocal Microscopy, Plasmid Preparation, Western Blot, Construct, Variant Assay

Journal: Cell Death & Disease

Article Title: Human VRK2 modulates apoptosis by interaction with Bcl-xL and regulation of BAX gene expression

doi: 10.1038/cddis.2013.40

Figure Lengend Snippet: Low levels of VRK2 facilitated release of cytochrome c and PARP processing. ( a ) VRK2 knockdown with si-VRK2-06 and si-Control (si-Ct) in three cell lines, lung carcinomas A549 and H1299 and cervical carcinoma HeLa. The reduction in endogenous protein level and its quantification 4 days after transfection are shown. Endogenous VRK2 protein was detected with a rabbit polyclonal antibody. ( b ) The release of cytochrome c was determined by its detection in cytosolic and mitochondrial fractions in extracts from HeLa cells prepared 96 h after siRNA transfection. The effect of si-VRK2-06 is shown in the top gel panel and quantification of cytochrome c distribution in the two fractions, represented as percentage of band intensity respect to the corresponding marker, is shown at the bottom. Actin and cytochrome c oxidase II were used as markers for the cytosolic and mitochondrial fractions respectively. ( c ) Effect of VRK2 knockdown on processing of PARP, a caspase target, in HeLa cells. The proteolytic processing of PARP was determined in a western blot and quantification of the 115 and 85-kDa bands, represented as percentage of band intensity, i s shown at the bottom. Immunoblots show a representative experiment and quantifications show the mean and S.D. from three independent experiments

Article Snippet: Rabbit polyclonal anti-Bax was from Abcam (Cambridge, UK).

Techniques: Knockdown, Control, Transfection, Marker, Western Blot

Journal: Scientific Reports

Article Title: Kaiso protects human umbilical vein endothelial cells against apoptosis by differentially regulating the expression of B-cell CLL/lymphoma 2 family members

doi: 10.1038/s41598-017-07559-0

Figure Lengend Snippet: P120ctn participates in the gene regulation of BCL2, BAX and BIK by Kaiso. HUVECs were transfected with pCDNA3.1-Kaiso for 48 h and stained with polyclonal rabbit anti-Kaiso (H-154) antibody (in red color) and monoclonal mouse anti-p120ctn antibody (in green color) ( A ). Cell nuclei were stained with DAPI ( A ,a). Arrowheads show the particle like structures where both Kaiso and p120ctn were observed ( A ,d). Scale bar represents 10 μm. HUVECs and HMEC-1s were co-transfected with pCMV-flag-Kaiso and pCMV-myc-p120ctn, and 48 h later the interaction of Kaiso and p120ctn was evaluated by co-immunoprecipitation using monoclonal mouse anti-FLAG antibody and monoclonal mouse anti-Myc tag antibody respectively ( B ). HUVECs were transfected with NC-siRNA, p120ctn-siRNA, pCDNA3.1 and pCDNA3.1-p120ctn respectively, and p120ctn expression was evaluated by Western blot 48 h after transfection ( C ). GAPDH served as a loading control. Values are presented as mean ± SD, *p < 0.01, compared with blank, n = 3. HUVECs were transfected with recombinant firefly luciferase reporter constructs pGL3b-BCL2 ( D ,a), pGL4.1-BAX and pGL4.1-BIK ( D ,b), together with pCDNA3.1, pCDNA3.1-Kaiso, pCDNA3.1-Kaiso and p120ctn-siRNA, pCDNA3.1-Kaiso and pCDNA3.1-p120ctn, and pCDNA3.1-p120ctn, respectively. Renilla luciferase reporter pRL-TK was used as an internal control. The untreated HUVECs served as a blank. Relative luciferase activity is presented as mean ± SD. *p < 0.01, n = 3.

Article Snippet: Antibodies used were monoclonal rabbit anti-Caspase-3 antibody and polyclonal rabbit anti-cleaved-Caspase-3 antibody (Cell Signaling Technology, Boston, MA), monoclonal mouse anti-Kaiso 6F/ 6F8-CHIP grade antibody (Abcam, Cambridge, UK), polyclonal rabbit anti-BCL2 antibody (Proteintech, Chicago, IL), polyclonal rabbit anti-BAX antibody (Proteintech), monoclonal mouse anti-BIK antibody (Cell Signaling Technology), monoclonal mouse anti-p120 Catenin antibody (BD Biosciences, San Jose, CA), monoclonal rabbit anti-GAPDH antibody (Cell Signaling Technology) (loading control), polyclonal rabbit anti-Histone H3 antibody (Proteintech) (loading control), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Proteintech) and HRP-conjugated goat anti-rabbit IgG (Proteintech).

Techniques: Transfection, Staining, Immunoprecipitation, Expressing, Western Blot, Control, Recombinant, Luciferase, Construct, Activity Assay

Journal:

Article Title: Transforming Growth Factor ?-Dependent Sequential Activation of Smad, Bim, and Caspase-9 Mediates Physiological Apoptosis in Gastric Epithelial Cells †

doi: 10.1128/MCB.25.22.10017-10028.2005

Figure Lengend Snippet: TGF-β1 induces a conformational change of Bax and expression of Bim and Bmf in SNU16 cells. (A) The heavy-membrane fraction from SNU16 cells treated with 10 ng/ml TGF-β1 for the period indicated was subjected to Western blotting to examine the expression of antiapoptotic molecules, Bcl-2, Bcl-XL, and Mcl-1 (Mcl-1 was below the level of detection). (B) SNU16 cells were treated with 10 ng/ml TGF-β1 for the period indicated and then subjected to Western blotting with anti-Bax antibody (top panel). To analyze the activation of Bax, immunoprecipitation was carried out with a specific antibody for the active form of Bax. The immunoprecipitated Bax was detected by Western blotting using a polyclonal antibody that binds all forms of Bax (middle panel). Oligomerization of Bax was examined by Western blotting with an anti-Bax antibody after incubation of cell lysates with the chemical cross-linker BMH (bottom panel). Stars indicate nonspecific bands. (C) SNU16 cells were treated with 10 ng/ml TGF-β1 for the period indicated and then subjected to Western blotting with antibodies to the BH3-only proteins indicated and actin. (D) Total RNA was extracted from SNU16 cell treated with 10 ng/ml TGF- β1 for the indicated times and analyzed by real-time PCR using specific primers for human BimEL sequence (forward primer, 5′-GCCCCTACCTCCCTACAGAC-3′; reverse primer, 5′-AAGATGAAAAGCGGGGATCT-3′). The expression level of Bim mRNA was normalized to that of GAPDH and is expressed as the ratio to the expression level of TGF-β1-untreated cells. The data shown represent the means ± SD (n = 3). (E) The indicated 3.9-kb nucleotide fragment of human bim gene (including the 5′ flanking region, exon 1, intron 1, and exon 2) was isolated by PCR, and the nucleotide fragment was cloned into a pBV-B2 vector upstream of the firefly luciferase gene. The transcription start site is numbered +1. (F) SNU16 cells transfected with the human bim reporter construct and the renilla luciferase expression construct pRT-TK were treated with 10 ng/ml TGF-β1 for 12 h, after which a dual-luciferase assay was performed. The relative luciferase activity of firefly was determined after normalization to the activity of renilla. The data shown represent the means ± SD (n = 3). cont, control. (G) SNU16 cells were transfected with an expression vector for Smad7 or an empty vector, and the dual-luciferase activity was analyzed as described in for panel F. The data shown represent the means ± SD (n = 3).

Article Snippet: Cell lysates were immunoprecipitated with anti-Bax monoclonal antibody (6A7 BD; PharMingen), and immunoprecipitates were then subjected to Western blotting with anti-Bax polyclonal antibody (Cell Signaling).

Techniques: Expressing, Western Blot, Activation Assay, Immunoprecipitation, Incubation, Real-time Polymerase Chain Reaction, Sequencing, Isolation, Clone Assay, Plasmid Preparation, Luciferase, Transfection, Construct, Activity Assay