Western ip cell lysis buffer

Following the instructions of the BCA protein analysis kit (Thermo Fisher Scientific, Waltham, MA, USA), GC cells were lysed in WESTERN & IP cell lysis buffer (Beyotime, Shanghai, China) and PMSF (AMRESCO, Solon, Ohio, USA). The supernatant was collected after centrifugation at 4°C for 10 min. The same amount of sample proteins was added to the wells for electrophoresis, and then transferred to the membrane by using 0.45 μm PVDF membrane (Amersham Hybond, GE Healthcare, Munich, Germany). Wells were incubated with rabbit antibody at 4°C overnight using anti-Rab7 (1-2000, Abcam, UK), PI3K (1-1000, Cell Signaling Technology, USA), pPI3K (1-1000, cell signal), AKT (1-1000, cell signal), pAKT (Ser308) (1-1000, Cell Signaling Technology, USA), and pAKT (Ser473) (1-1000, Cell Signaling Technology, USA). After washing for 10 min in TBST, the primary antibody was incubated with enhanced chemiluminescence substrate (Abcam, UK) for 2 h at room temperature, and then protein signal detection was performed using enhanced chemiluminescence (Lulong Biotech, Xiamen, China).

The heart tissue was lysed with Western & IP cell lysis buffer (Beyotime, Shanghai, China) supplemented with phosphatase inhibitors and PMSF (Amresco, Solon, Ohio, USA) for 30 min on ice, centrifuged at 12,000 g for 15 min at 4°C, supernatants collected and total protein was measured using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein were separated on 10% sodium dodecyl sulfate polyacrylamide gel and then transferred onto a 0.45μm PVDF membrane (AmershamHybond, GE Healthcare, München, Germany). Membranes were then blocked with TBST containing 0.5% bovine serum albumin (BSA; Amresco, Solon, Ohio, USA) for 2 h at room temperature and then incubated with the primary antibodies: rabbit anti-TLR4 (1:1000 dilution; Abcam, USA), rabbit anti-NF-κb (1:1000 dilution; CST, USA), rabbit anti-Bax (1:1000 dilution; CST, USA), rabbit anti-Bcl-2 (1:1000 dilution; CST, USA), rabbit anti-PI3K (1:1000 dilution; Abcam, USA), rabbit anti-p-AKT (1:1000 dilution; Abcam, USA), rabbit anti-GAPDH (1:5000 dilution; CST, USA) overnight at 4°C. After washing with TBST, membranes were incubated with goat anti-rabbit secondary antibody at room temperature for 1 h and washed with TBST. Target proteins were detected using ECL kits (Thermo Fisher Scientific). The protein levels were analyzed with Image J. GAPDH protein expression was used as an internal control.