Anti cleaved caspase 3 asp175 9664

Rapamycin (R0395), cycloheximide (CHX, A6185), AKT Inhibitor (A6730), triciribine (t3830), 3-MA (M9281), anti-β-actin antibody (A3853), and anti-LC3 antibody (L8918) were obtained from Sigma-Aldrich (Shanghai, China). Anti-AKT, anti-phospho-AKT, anti-ATG5 (6230), and anti-cleaved caspase 3 (Asp175) (9664) antibodies were obtained from Cell Signaling. The anti-gE antibody and anti-US3 antibody were produced from immunized mice. FITC-conjugated goat anti-mouse secondary antibodies and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit secondary antibodies were purchased from Zhongshan Jinqiao, China. The gene for US3 was amplified using the primers listed in Table S1 and cloned into the pCAGGS vector (Addgene, USA) and the pDsRed-Express-N1 vector (BD Biosciences Clontech, USA). For kinase-dead US3, we generated several point mutation mutants, including a lysine to glycine substitution at position 136 (K136G) and an aspartate to alanine substitution at position 223 (D223A), and a combination of these two positions. The primers are listed in Table S1. The nucleotide sequences of the plasmids encoding US3 and kinase-dead US3 were confirmed to ensure that the correct clones were used in the study. Plasmid GFP-LC3 was kindly provided by Professor William Jackson.