Western blotting were performed as we described previously [27 (link)]. Cells were lysed in 2× Laemmli’s sample buffer (Bio-rad, CA, USA) for 20 min. on ice. After boiling for 10 min. at 95°C, equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred at 100 V for 1 hr onto a PVDF membrane (Millipore, USA). The membrane was blocked and incubated with primary antibodies against phospho-eNOS (1:1000), eNOS (1:2000), phospho-Akt (1:1000), Akt (1:2000), phospho-Erk1/2 (1:1000), Erk1/2 (1:2000), Atg7 (1:1000), p62 (1:5000), LC3B (1:4000), or β-actin (1:5000) overnight at 4°C. Antibodies against p62 were purchased from Abcam Inc. (UK) and all others were purchased from Cell Signaling Technology Inc. (MA, USA). Following primary antibody incubation, the membrane was further incubated with 1:15000 secondary antibodies IRDye 800CW donkey anti-rabbit IgG and 680RD goat anti-mouse IgG (Licor, NE, USA). The immunoreactive bands were detected by Licor Odyssey Fc imaging system. Densitometric analysis of images was performed using Licor Image Studio software (NE, USA).