Sds page sample buffer

Cells were harvested in LIPA buffer (Sigma) as previously reported^{13 (link)}. After centrifugation at 15,000 × g for 5 min, the supernatant was boiled for 5 min in SDS-PAGE sample buffer (Wako) and loaded onto 5–20% gradient or 15% gels for 1~2 h. Proteins were transferred to nitrocellulose membranes (Bio-Rad) by using the Trans-Blot Electrophoretic Transfer cell (Bio-Rad). The membrane was washed in 20 mM Tris-HCl buffer with 1% Tween 20 (TBS-T) (Sigma-Aldrich), incubated for 1 h in blocking buffer (5% milk powder (Merck) in TBS-T), and then further incubated overnight with primary antibodies; Anti-LC3 (Cell signaling) and anti-GAPDH (Santa Cruz) antibodies were purchased and diluted in blocking buffer by 1:3000. The horseradish peroxidase (HRP)-labeled secondary antibody (Dako) was diluted by 1:5000 in TBS-T for secondary antibody staining for 1 h at RT. The membrane was then washed in TBS-T and the signals were developed using the Lumi-Light reagent (Roche) for 5 min. The signal was detected on the LAS-3000 imaging system (Fuji).