| U2OS cells were lysed in 10 mM KPO4, 1 mM EDTA, 10 mM MgCl2, 5 mM EGTA, 50 mM bis-glycerophosphate, 0.5% NP40, 0.1% Brij35, 0.1% sodium deoxycholate, 1 mM NaVO4, 5 mM NaF, 2 mM DTT, and complete protease inhibitors (Sigma, P8340-5ML). |
Proteins were resolved by SDS-PAGE, transferred to PVDF (for low molecular weight proteins like LC3) or nitrocellulose membranes, blocked with 4% w/v non-fat dry milk in 1x TBS-T (TBS with 0.05% Tween 20), and probed with primary antibodies in blocking buffer at 4°C overnight followed by secondary antibodies in blocking buffer for 45 minutes at room temperature. |
Proteins were detected by chemiluminescence using ECL (100 nM Tris, pH 6.8, luminol (1.25 mM), p-coumaric acid (198 μM), H2O2 (0.009%)) or SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher, 34095). |