Anti bcl 2

Cells or tumors samples were lysed with RIPA buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris pH 8.0, 1% sodium deoxycholate, 1% NP-40, 0.5% SDS) supplemented with 1 mM dithiothreitol and protease inhibitors. Cell lysates were then separated by SDS-PAGE and analyzed by standard western blotting protocol. Antibodies used were anti-BCL-X_L (Cell Signaling, 54H6, 1:1000), anti-MCL-1 (Cell Signaling, D35A5, 1:1000), a nti-BCL-2 (Cell Signaling, 50E3, 1:500), and anti-β-actin (Cell Signaling, 1:10,000).

Protein lysates were separated by 10% SDS-PAGE and then transferred to nitrocellulose membranes by electroblotting. The membranes were incubated with anti-IκB kinase, anti-GADPH, anti-BCL2, anti-BAX, anti-p21, or anti-β-ACTIN antibodies (Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C before subsequent incubation with secondary antibodies conjugated with horseradish peroxidase for 1 h at 37 °C. Proteins were visualized using enhanced chemiluminescence reagent.

Total protein extraction from lung tissues was conducted using radioimmunoprecipitation assay lysis buffer with PMSF. Total protein was quantified by BCA assay. Protein samples were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels, and transferred to polyvinylidene fluoride membranes. After 1 h of blocking with 5% dry skimmed milk at room temperature, membranes were probed with the following primary antibodies at a dilution of 1:1000: anti-prosurfactant protein B (SPB; Santa Cruz, Dalla, TX), anti-prosurfactant protein C (SPC; Abcam, Cambridge, UK), anti-FoxM1 (Proteintech, Rosemont, IL), anti-β-actin (DEWEIBIO, China), anti-β-catenin, anti-VE-cadherin, anti-BCL-2, and anti-BAX (Cell Signaling Technology, Danvers, MA) at 4 °C overnight. After washing three times with Tris-buffered saline containing Tween, membranes were incubated for 1 h using horseradish peroxidase-conjugated secondary antibodies at room temperature. The loading control was β-actin. Imaging was performed using a gel imager and evaluated using ImageJ software v1.4.0.

Western blotting was performed as described previously [62 (link)]. The primary antibodies used in this study include anti-γH2A.X (1:1000, 2577S, Cell Signal Technology, Danvers, MA, USA), anti-p53 (1:500, sc-6243, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p53ser15 (1:1000, 9286S, Cell Signal Technology, Danvers, MA, USA), anti-cleaved PARP1 (1:1000, 5625S, Cell Signal Technology, Danvers, MA, USA), anti-MCL1 (1:1000, 39224S, Cell Signal Technology, Danvers, MA, USA), anti-BCL2 (1:1000, 2872S, Cell Signal Technology, Danvers, MA, USA), anti-ACTIN (1:150,000, MAB1501, Millipore, Burlington, MA, USA). The secondary antibodies used were HRP-linked anti-rabbit IgG (1:4000, 7074S, Cell Signaling Technology, Danvers, MA, USA) and HRP-linked anti-mouse IgG (1:4000, 7076S, Cell Signaling Technology, Danvers, MA, USA).

Catalog numbers and dilution fold are listed in parentheses. The following antibodies were purchased from Cell Signaling Technology: Anti-Bak (3792; 1:1000), anti-Bax (2772; 1:1000), anti-Bcl-2 (2870; 1:1000), anti-Bcl-xL (2764; 1:500), anti-CHK1 (2360; 1:1000), anti-phospho-CHK1 (S345; 2348; 1:1000), anti-cleaved caspase-3 (9661; 1:1000), anti-cleaved caspase-7 (9491; 1:1000), anti-cleaved caspase-8 (9496; 1:500), anti-cleaved caspase-9 (9501; 1:1000), anti-cytochrome c (4272; 1:500), anti-H2A.X (7631; 1:1000), anti-phospho-H2A.X (S139; 9718; 1:1000), anti-p53 (2524; 1:1000), anti-p53 (2527; 1:1000), anti-phospho-p53 (S6; 9285; 1:1000), anti-phospho-p53 (S15; 9284; 1:1000), anti-p21 (2946; 1:1000), and anti-voltage-dependent anion channel (VDAC; 4866; 1:1000). The following antibodies were purchased from Santa Cruz Biotechnology: Anti-CD95 (1023; 1:500), anti-cyclophilin 40, also known as anti-CYPD (137157; 1:400), anti-His tag (803; 1:500), anti-lamin A (20680; 1:1000), anti-MDM2 (965; 1:500), and anti-ubiquitin (8017; 1:200). Anti-PEPD (Ab86507; 1:500) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374; 1:5000) were purchased from Abcam and EMD Millipore, respectively. Anti-mouse IgG-horseradish peroxidase (IgG-HRP; NA931V; 1:4000–5000) and anti-rabbit IgG-HRP (NA934V; 1:5000) were purchased from GE Healthcare.