Western blot infrared imaging system

Western blot was performed as described previously.^{19 (link)} Briefly, proteins obtained from lysed cells were denatured and loaded on SDS polyacrylamide gel. Afterwards, the proteins were transferred onto PVDF membranes, blocked in TBS-T (150 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.5, and 0.1% Tween-20) containing 5% (w/v) bovine serum albumin, and incubated with the corresponding primary and secondary antibodies. The specific bands were analyzed by the western blot infrared imaging system (LI-COR Biosciences). For immunoprecipitation, cells were lysed with nonidet P40 lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM DTT, 10% glycerol) containing protease inhibitors. After centrifugation, the supernatants were incubated with antibody overnight and then Protein A/G agarose for 2 h at 4 °C. Immunocomplexes were washed and analyzed by western blotting.

For immunoprecipitation, cells were lysed with NP40 lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM DTT, 10% glycerol) containing protease inhibitors. After centrifugation, the supernatants were incubated with antibody overnight and then Protein A/G agarose for 2 h at 4 °C. Immunocomplexes were washed and analyzed by Western blot. For Western blot, the proteins from lysed cells were denatured and separated with SDS-PAGE. Then, the proteins were transferred to PVDF membranes, blocked and incubated with the corresponding primary and secondary antibodies. The specific bands were analyzed by the Western blot infrared imaging system (LI-COR Biosciences). The following antibodies were used: EVA1A: A8070, ABclonal; CHOP: 2895, Cell signaling Technology (CST); IRE1: 14C10, CST; ATF6: 65880 T, CST; Actin: AC026, ABclonal; Flag: 20543-1-AP, Proteintech; Caspase 8: 4927, CST; C-Caspase 9: 9509, CST; C-Caspase 3: 9664, CST; Caspase 12: 2202, CST; C-Parp: 9548, CST; GFP: ab290, Abcam; MCL1: 94296, CST; Bak: 12105, CST; LC3: 27543,Sigma; P62: 18420-1-AP, Proteintech; BNIP3: ab10433, Abcam; TIM23: 111263-A, Proteintech; TOM20: ab56783, Abcam.