Biochemical Profiling of Autophagy Pathways

RL71 (>97% purity, HPLC) was synthesized as described previously.^{14 (link)} anti-LC3B, anti-ubiqutin, anti-CHOP, anti-PARP, anti-p-JNK, anti-JNK, anti-p-PERK (Thr980), anti-p-AMPK (Thr172), anti-p-mTOR (Ser2448, 2971), anti-p-p70S6K (Thr389), anti-Cox4 and anti-cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-Grp78, anti-ATF4, anti-ATF6, anti-β-actin and anti-GFP antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-SQSTM1/p62, anti-ATG5 and anti-ATG7 antibodies were from Abcam (Cambridge, UK) and anti-LC3 was from Novus Biologicals (Littleton, CO, USA). GFP-LC3 plasmid was purchased from Yingrun Biotechnologies Inc. (Changsha, China). Compound C (CC) and BAPTA were obtained from Calbiochem (San Diego, CA, USA). zVAD was from Selleck Chemicals (Houston, TX, USA). Chloroquine (CQ), STO-609, SP600125 and PBA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ER-specific dye ER tracker Red, the mitochondrial specific dye MitoTraker Red CMXRos (M7512), JC-1 (T-3168), Fura-2/AM and Lipofectamine 3000 transfection reagent were purchased from Life Technologies (Grand Island, NY, USA). The Ca²⁺-ATPase activity assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing). All of the other chemicals were purchased from Sigma-Aldrich.