Anti card11

Cell pellets were re-suspended in RIPA buffer and quantified using the BCA assay (Thermo Scientific Pierce BCA Protein Assay Kit). Equal amounts of protein were run on an SDS-PAGE gel (8%−12%), then transferred to a nitrocellulose membrane and incubated in 5% milk in TBS-T for one hour, followed by incubation in primary antibodies overnight. Blots were then washed with TBS-T three times for 20 minutes each, then incubated in HRP conjugated secondary antibodies (GE Healthcare, 1:2000) for one hour. HRP was visualized using an enhanced chemiluminescence detection kit (PerkinElmer). The following antibodies were used: anti-CARD11 (Cell Signaling Technology Cat# 4435, RRID:AB_2070359; Abcam Cat# ab113409, RRID:AB_10861854; 1:1000), anti-phospho S6 (Cell Signaling Technology Cat# 2215, RRID:AB_331682; 1:1000), anti-S6 (Cell Signaling Technology Cat# 2217, RRID:AB_331355; 1:3000), anti-GAPDH (Sigma-Aldrich Cat# G8795, RRID:AB_1078991: 1:5000), anti-LC3B (Cell Signaling Technology Cat# 2775, RRID:AB_915950; 1:1000), anti-Alpha Tubulin (Sigma-Aldrich Cat# T9026, RRID:AB_477593; 1:3000) and anti-Beta Actin (Sigma-Aldrich Cat# A1978, RRID:AB_476692; 1:3000).