Anti ha clone 3f10

Total cell lysates were prepared by sonicating and boiling cell pellets in 1X Laemmli reducing sample buffer. Detergent-soluble cell lysates were prepared by lysing cells in cold isotonic lysis buffer (10 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, along with protease inhibitor cocktail and 1 mM PMSF for 15 minutes on ice and centrifuged for 10 minutes at 20,000 g. The clarified supernatant was used as the detergent soluble cell fraction. Lysates were electrophoresed (SDS-PAGE gels) and transferred to nitrocellulose membranes (Whatmann). Proteins were detected by immunoblotting.
Antibodies used in this study were purchased from the following sources: anti-actin and FLAG (Sigma-Aldrich); anti-ubiquitin clone P4D1, goat, anti-rabbit/mouse/rat IgG-conjugated horseradish peroxidase, p21, ERK and Mcl-1 (Santa Cruz Biotechnology); USP7, USP5, BRAF and p73 (Bethyl Laboratories); anti–poly(ADP-ribose) polymerase (PARP), pERK, Caspase8, Caspase3, Bid, Bax, Bak (Cell Signaling Technology); anti-HA (clone 3F10; Roche Applied Science); CD95/Fas (Clone EPR5700; Epitomics); anti-NOXA, p53 (DO-1; CalBiochem); anti-DR4, DR5 (ProSci incorporated) and FAS monoclonal antibody (CH11; Millipore).

Total proteins were extracted from GC tissues or cells using RIPA lysis buffer containing protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). The lysates were mixed with SDS loading buffer, boiled for 8 min, resolved by SDS-PAGE, and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies: anti-LDHA (#3582S, Cell Signaling Technology), anti-HA (clone 3F10, #11867423001, Roche), anti-FLAG M2 (#F1804; Sigma), anti-His TAG (#12698S, Cell Signaling Technology), pan succinyl-lysine antibody (#PTM-401; PTM Bio, Hangzhou, China), anti-β-actin (#4970; Cell Signaling Technology), or anti-CPT1A antibody (#12252; Cell Signaling Technology).

The cell lysates were prepared in lysis buffer (100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% Triton X-100) at 4 °C, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The following proteins were detected using specific antibodies: Anti-HA (clone 3F10, Roche, 1:5000 dilution), Anti-α-actinin (H-2, sc-17829, Santa Cruz Biotechnology, 1:5000 dilution), Anti-AGT (11992-1-AP, Proteintech Group, 1:1000 dilution), Anti-GR (H-300, sc-8992, Santa Cruz Biotechnology, 1:1000 dilution), and Anti-p53 (DO-1, sc-126, Santa Cruz Biotechnology, 1:1000 dilution).

Cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin. Protein concentration was measured by BCA Protein Assay (Pierce). Total protein (10 μg) was resolved on 10% SDS-PAGE, transferred to PVDF membranes (Immobilon-P, Millipore), and probed with primary antibodies against MTA2 (sc-55566), HDAC1 (sc-7872), SIRT1 (sc-15404) and p53 (sc-126) (Santa Cruz); YY1 (#2185), p21 (#2946), PUMA (#4976), CDK4 (#2906), CDK6 (#3136) and FAS (#8023) (Cell Signaling); MDM4 (mAb 8C6) and MDM2 (mAb 2A10) (EMD Bioscience); or anti-HA (clone 3F10, Roche), followed by incubation with horseradish peroxidase-(HRP) conjugated sheep anti-mouse or anti-rabbit Ig (Amersham). Signals were developed using SuperSignal West Femto (Pierce). Membranes were stripped and reprobed using α-tubulin mouse mAb (B-5-1-2, Sigma) or β-actin (JLA20, Millipore). Immunoblots were quantified by densitometry using VersaDoc MP 4000 (Bio-Rad).