Caspase 3 9664s

Whole-cell lysates from cells and tissues were isolated using RIPA cell lysis buffer with protease inhibitors (MCE). Protein concentration was assayed using Pierce^TM BCA protein assay (23225, Thermo Fisher Scientific). Primary antibodies were incubated overnight at 4 °C, and specific proteins were visualized by Pierce^TM ECL plus (32109, Thermo Fisher Scientific). Band intensities were measured using Image (Tannon, Shanghai) and normalized to β-actin. Primary antibodies used in this study are as follows: PDIA4 (14712-1-AP, Proteintech), Caspase-3 (9664S, Cell Signaling Technology), Necroptosis antibody sampler kit (98110T, Cell Signaling Technology), LC3 (M152-3, MBL Life Science), SQSTM1 (ab56416, Abcam), ATG5 (ab108327, Abcam), GRP78 (66574-1-Ig, Proteintech), PERK (24390-1-AP, Proteintech), p-PERK (29546-1-AP, Proteintech), GPX4 (ab125066, Abcam), ATF4 (10835-1-AP, Proteintech), SLC7A11 (26864-1-AP, Proteintech), and β-actin (sc-47778, Santa Cruz).
For the immunoprecipitation assay, 786-O cells that overexpress PDIA4 were collected in RIPA lysis buffer. Supernatants were immune-precipitated with 2 μg PDIA4 antibody overnight and then incubated with protein G agarose beads for another 5 h at 4 °C. SQSTM1 were then detected as described for western blot analyses.

Antibodies against LC3B (3868S), Ub (3936S), and active Caspase-3 (9664S) were purchased from Cell Signaling. Antibody against β-actin (sc-8432) was obtained from Santa Cruz. Antibody against RACK1 (610178) was from BD Biosciences. Antibody against electron-transferring-flavoprotein dehydrogenase (ETFDH) (A6585) was from ABclonal Biotech. Antibody against very long-chain acyl-CoA dehydrogenase (VLCAD) (14527-1-AP) was from Proteintech. Total protein extracts were prepared and dissolved in SDS sample buffer. Protein extracts were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were probed with antibodies and visualized with an electrochemiluminescence kit (Amersham).