P21 waf1 cip1

Cells were lysed on ice using the Pro-prep™ protein extraction kit (Intron Biotechnology, Korea), and expression of cell cycle mediators and autophagy related signaling molecules were evaluated using western blots. Briefly, 20 μg of protein lysates was resolved by 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Invitrogen). The membrane was blocked for 1 h in PBS containing 5% BSA and 0.2% Tween-20 and incubated overnight with primary antibodies. The following primary antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA): Cyclin D1 (Cat# 2978, 1:1000 dilution), P27/Kip1 (Cat# 3686, 1:1000), P21 Waf1/Cip1 (Cat# 2947, 1:1000), AMPKα (Cat#5832, 1:1000), Phospho-AMPKα (Cat#50081, 1:500), ULK1 (Cat#8054, 1:500), Phospho-ULK1(Ser555, Cat#5869, 1:500), while antibodies to LC3B were purchased from Invitrogen (Cat# PA1–16930, 1:1000). Antibody-reactive proteins were detected by means of the appropriate HRP-conjugated secondary antibodies and an enhanced chemiluminescent substrate (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific). Immunostained bands were analyzed using ChemiDoc MP (Bio-Rad) and relative quantitation was done after normalization to GAPDH band in same blots.