Anti ampkα

Protein sample preparation and Western immunoblotting were performed as previously described (Qin et al., 2011 (link); Pandey et al., 2017 (link)). Primary antibodies used in the immunoblotting analysis included anti-p-ULK1, anti-ULK1, anti-ASK1, anti-p-ASK1, anti-SAPK/JNK, anti-p-SAPK/JNK (Cell signaling); anti-AMPKα, anti-p-AMPKα, anti-Atg9a (Thermo Scientific); anti-LC3, anti-p-ASK1 and anti-GAPDH (Santa Cruz Biotech., Inc); anti-BAK, anti-BAX (EMD Millipore), anti-p-IRE1α (GeneTex, Inc), anti-IRE1α (Novus Biologicals). Dilution of primary antibodies was 1:1,000. Secondary antibody HRP anti-IgG (Sigma-Aldrich, USA, 1:1,000~5,000) was used in the immunoblotting analysis. Densitometry of blots was performed using the ImageJ software package (http://rsbweb.nih.gov/ij/). The relative expression levels of target proteins and the ratio of blot LC3-II/LC3-I at the indicated time points were calculated as previously described (Qin et al., 2011 (link)). All Westerns were performed in triplicate and representative images are shown.

Whole cell protein extracts were prepared according to Lewinska et al. [16 (link)]. Polyvinylidene difluoride (PVDF) membranes were incubated with the primary antibodies anti-p21 (1:100), anti-p53 (1:500), anti-p27 (1:200), anti-GLUT1 (1:1000), anti-HK2 (1:200), anti-PKM2 (1:1000), anti-LDHA (1:1000), anti-phospho-AMPKα (Thr172) (1:750), anti-AMPKα (1:1000), anti-phospho-AKT (Ser473) (1:1750), anti-AKT (1:1000) or anti-β-actin (1:1000) (Thermo Fisher Scientific, Santa Cruz, Abcam, Cell Signaling) and a secondary antibody conjugated to HRP (1:50,000, Sigma–Aldrich). The respective proteins were detected using a Clarity™ Western ECL Blotting Substrate (BioRad) and a G:BOX imaging system (Syngene, Cambridge, UK) according to the manufacturer’s instructions. Densitometry measurements of the bands were performed using GelQuantNET software (http://biochemlabsolutions.com/GelQuantNET.html). The data represent the relative density normalized to β-actin.