Gapdh 14c10

Cells lysates were harvested by scraping and suspension in lysis buffer (62.5 mM Tris HCl/pH 6.8, 10% glycerol and 2% SDS), heated and sonicated. The protein concentrations of the cell lysates were estimated using a Pierce^® BCA Protein Assay kit. Equal quantities of protein were loaded onto and separated by SDS–polyacrylamide gels (4–20% Mini-PROTEAN^® TGX™ Gel, BioRad). The separated proteins were transferred and immobilized onto Amersham™ nitrocellulose membranes (GE Healthcare Life Science). Primary antibodies against p53 (DO-7) (#M7001, Dako), MDM2 (Ab-1) (#OP46, Merck Millipore), p21^WAF1 (EA10) (#OP64, Calbiochem), p-ERK (E-4) (sc-7383, Santa Cruz), ERK (K-23) (sc-94, Santa Cruz), GAPDH (14C10) (#2118, Cell Signaling Technology), BRAF^V600E (VE1, Spring Bioscience), Actin (A4700, Sigma) and secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (#P0447/P0448, Dako) were used. All antibodies were diluted in 5% (w/v) non-fat milk or BSA in TBS-tween (20 mM Tris/pH 6.8, 137 mM NaCl, 0.1% tween-20). Protein signals were visualized using enhanced chemiluminescence (GE Healthcare Life Sciences) and X-ray film (Fujifilm).

The lysates were loaded on SDS-PAGE gels, followed by blotting onto polyvinylidene difluoride membranes (BioRad Laboratories, Inc., CA, USA). The following antibodies were obtained from Cell Signaling Technology (MA, USA): GAPDH (14C10) (cat # 2118, 1:1000 dilution), Lamin B1 (D4Q4Z) (cat# 12586, 1:1000 dilution) (Cell Signaling Technology), IGF1 Receptor β (D23H3) XP® (cat # 9750, 1:1000 dilution), Phospho-IGF1 Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) (cat # 3021, 1:1000 dilution), AKT (cat # 9272, 1:1000 dilution), Phospho-AKT (Ser473) (cat # 9271, 1:1000 dilution). The antibodies ERK1/ERK2 (cat # MAB1576, 0.5 µg/mL dilution) and Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) (cat # MAB1018, 0.5 µg/mL dilution) were obtained from R&D Systems Inc. (MN, USA). The anti-BCOR antibody (cat# ab88112, 0.5 µg/mL dilution) was purchased from Abcam (Cambridge, UK). The HRP-linked secondary antibodies were obtained from Cell Signaling Technology (MA, USA) (anti-mouse IgG (cat# 7076)) and Sera Care (MA, USA) (anti-rabbit IgG (cat# 074-1516)). Detection was done by SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific, MA, USA), and imaging was performed on Fusion Pulse TS (Vilber Lourmat, Eberhardzell, Germany).

Lung tissues were homogenized in ice-cold lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail (Sigma-Aldrich, USA) for 40 min, then centrifuged at 12,000 g for 20 min. Protein concentrations were measured using the BCA assay. 30 μg of total protein was sampled and separated in 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). The membranes were then blocked with 5% fat-free milk in Tris-buffered saline and 0.5% Tween-20 (TBS-T) for 2 h and then incubated with corresponding primary antibodies overnight at 4 °C. After washing with TBS-T, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (ZB2306) for 1 h at room temperature. Finally, signals were gained by using the enhanced chemiluminescence (ECL) kit (CW Biotechnology, Beijing, China) and were exposed by using a biological molecular imaging instrument (LAS4000mini, JAPAN). The immunoblots intensities were quantified using Quantity One software (Bio-Rad, CA, USA). The primary antibodies including P38 (D13E1), p-P38 (D3F9), ERK (137F5), P-ERK (D13.14.4E), JNK (56G8), p-JNK (98F2), β-Tublin (9F3) and GAPDH (14C10) were purchased from Cell Signaling Technology, Inc. (CST, MA, USA).