Anti lc3 2 1

Human gastric cancer cell lines, including BGC-823, MGC, SGC-790, HGC, and MKN-45 were purchased from the American Type Culture Collection in 2015. All cells were cultured in DMEM, supplemented with 10% fetal bovine serum, and were grown in a humidified atmosphere containing a 95% air, 5% CO2 mixture at 37°C. Anti-AM, anti-P-JNK, anti-JNK, anti-P-PKA, anti-PKA, and anti-LC3-II/I antibodies were obtained from Abcam Technology (Cambridge, MA). Anti-cleaved-caspase3, anti-caspase3, anti-P-AKT, anti-AKT, anti-Beclin, and anti-CRLR antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-GAPDH, anti-Bax, anti-Bcl2, anti-RAMP1, anti-RAMP2, and anti-RAMP3 antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Hepatopancreas was lysed in lysis RIPA buffer (Beyotime, China) for 30 min on ice. After incubation, lysates were centrifuged (14,000 × g, 15 min) at 4°C, and each supernatant was collected. Total protein was quantified using the traditional BCA method. Fifty micrograms of protein was separated by 15% sodium dodecyl sulfate–PAGE and electrotransferred onto polyvinylidene difluoride membrane (Bio-Rad). The membranes were incubated in 20 mM tris-buffered saline (TBS) containing 5% BSA for 1 h at room temperature. The membranes were washed with tris-buffered saline and tween 20 (TBST) for 5 min and incubated with the anti-LC3I/II (1:500, Abcam), beclin1 (1:1,000, CST), TOR (1:500, CST), p-TOR (1:500, CST), and actin (1:1,000, CST) at 4°C overnight in a shaking incubator and then washed with (3 × 5 min per wash), and the protein was detected using alkaline phosphatase (1:2,000) as the secondary antibody was incubated for 2 h at room temperature. The membrane was detected with 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium substrate (Sangon Biotech, Shanghai, China). To quantify the signals, gray intensities of Western blots were calculated using ImageJ⁷.