Any kd sds page gels

Expanded effector T cells (1×10⁶ per time point) deprived of IL-2 for 0–3 days were washed in cold PBS, and lysed in 1% Nonidet P-40 (NP-40) lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 1 mM Na₃VO₄, 1 mM NaF) containing complete protease inhibitors (Roche Diagnostics Corp., Indianapolis, IN, USA) for 30 min on ice. For LC3 blotting, cells were lysed in 10 mM Tris-Cl, pH 6.8, 69 mM NaCl, 0.5 mM EGTA, 0.5% Triton X-100, 5% glycerol containing complete protease inhibitors (Roche Diagnostics Corp.) for 30 min on ice. Cleared lysates were boiled in 2x reducing sample buffer and resolved on Any kD SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were transferred to nitrocellulose or PVDF (for LC3) on a Trans-Blot Turbo system (Bio-Rad), blocked with 2% Tropix I-Block (Thermo Fisher Scientific) or 6% milk (Bio-Rad) in TBS/0.1% Tween, and probed with the following Abs: anti-BIM (Enzo Life Sciences Inc); anti-PUMA (Cell Signaling Technology); anti-Bcl-xL, anti-Mcl-1, anti-Bcl-2 (BD Biosciences); anti-LC3B and anti-β-actin (Sigma-Aldrich). Bound Abs were detected using HRP-conjugated secondary Abs (Southern Biotech, Birmingham, AL USA; Agilent Technologies) and ECL (Thermo Fisher Scientific). Densitometry analysis was performed using ImageJ software (www.imagej.net). Full blots are included as Supplementary Information.