Anti c caspase3

Tissues were lysed using RIPA buffer (Beyotime, Shanghai, China), and lysates were loaded onto 12% bis–tris-acrylamide gels (Thermo Fisher, Waltham, MA, USA). The separated protein bands were electrotransferred onto polyvinylidene fluoride membranes (Millipore, Bradford, MA, USA), and then immersed in 5% nonfat dry milk (Solarbio, Beijing, China). After that, the membranes were incubated with anti-BCL2-associated x protein (anti-Bax) (1:5000; Abcam, Cambridge, UK), anti-matrix metalloprotein 2 (anti-MMP2) (1:5000; Abcam, Cambridge, UK), anti-MMP9 (1:3000; Abcam, Cambridge, UK), anti-caspase 3 (anti-t-caspase 3) (1:5000; Abcam, Cambridge, UK), anti-cleaved-caspase 3 (anti-C-caspase 3) (1:8000; Abcam, Cambridge, UK), anti-proliferating cell nuclear antigen (anti-PCNA) (1:3000; Abcam, Cambridge, UK), anti-MYC proto-oncogene, bHLH transcription factor (anti-c-Myc; 1:1000; Abcam, Cambridge, UK), anti-KRAS proto-oncogene, GTPase (anti-K-RAS; 1:1000; Abcam, Cambridge, UK), anti-B-Raf proto-oncogene, serine/threonine kinase (anti-BRAF; 1:2000; Abcam, Cambridge, UK) and anti-GAPDH (1:15,000; Abcam, Cambridge, UK), respectively. Then, the membranes were incubated with secondary antibody (1:8000; Abcam, Cambridge, UK). Finally, RapidStep ECL Reagent (Millipore, Bradford, MA, USA) was used to visualize the protein bands. GAPDH was chosen as a control.

Total protein was extracted from GC cells and tissues. The proteins were transferred to PVDF membranes (Millipore USA) after SDS-PAGE electrophoresis. After blocking in TBST buffer with 5% skimmed milk, the membranes were incubated with primary antibodies overnight at 4 °C. The membranes were rinsed three times with TBST and then incubated with secondary antibodies at room temperature for 2 h. After the membranes were washed with TBST three times, the bands were detected using an enhanced chemiluminescence detection system with Chemiluminescence HRP Substrate (Millipore, WBKL0100). Anti-Bcl2, anti-Bax, anti-caspase3, anti-c-caspase3, anti-β-actin, anti-P62, anti-Beclin1, anti-LC3, and anti-MTMR3 were purchased from Abcam (Cambridge, UK). Anti-rabbit IgG-HRP and anti-mouse IgG-HPR antibodies were obtained from Santa Cruz (Dallas, TX, USA).

Cells were lysed with RIPA buffer (Sangon), and proteins were quantified using a BSA Kit (Beyotime, Shanghai, China). Tumor proteins were extracted by a One-Step Animal Tissue Active Protein Extraction kit (Sangon). Protein (20 μg) was loaded and separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (Fdbio Science, Hangzhou, China) at 80 V for 30 min and then 120 V for 60 min followed by transfer to polyvinylidene fluoride (PVDF) membranes (Millipore) at 300 mA for 90 min. After blocking with 5% fat-free milk for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-Siglec-15 (1 : 1000, Santa Cruz), anti-GAPDH (1 : 5000, Sino Biological), anti-AGO2 (1 : 20, Abcam), anti-C-caspase3 (1 : 1000, Abcam), anti-caspase3 (1 : 1000, Abcam), and anti-Bcl-2 (1 : 1000, Abcam). After incubation with HRP-conjugated antibodies (1 : 5000, Sino Biological) for 2 h at room temperature, an ECL kit (Fdbio Science) was used for visualization of protein bands.