Western Blot Analysis of Protein Expression

This procedure was performed as in our previous report.^{50 (link)} Protein extracts from cells were prepared in RIPA buffer (Beyotime) containing 1% NP-40 (Beyotime) and a cocktail of protease and phosphatase inhibitors (Beyotime). Protein concentrations were determined using the Bio-Rad protein assay (Bio- Rad, Hercules, CA, USA; 500-0006). Thirty micrograms of total protein were loaded onto 8-10% SDS-PAGE gels and transferred onto a PVDF membrane using a Trans-Blot Turbo SystemTM (Bio-Rad) and Transfer packTM (Bio-Rad; 1704156). The primary antibodies used for the analysis were as follows: Tuj1 (#ab18207; 1:1000; Abcam, Cambridge, UK), GFAP (ab7260; 1:1000; Abcam), LC3 (#ab128025; 1:500; Abcam), Beclin-1(#ab302669; 1:1000; Abcam), p62 (#ab207305;1:1000; Abcam), PPARγ (#ab178860; 1: 1000; Abcam), and GAPDH (1:1000; Beyotime). Protein bands were visualized using HRP-linked secondary antibodies (Bio-Rad, anti-mouse 1706516, anti-rabbit 1706515) and the Clarity Western ECL substrate (Bio-Rad, 1705061) with a ChemiDoc MP imaging system (Bio-Rad). The blots were stripped with glycine (0.2 M, pH 2.5) stripping buffer and reprobed with the appropriate antibodies. The intensity of each band was detected and measured by Image J software (version 1.4; National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/ij/).