Anti t eif2α

OPCs were washed twice with PBS and lysed in ice-cold RIPA buffer (Sigma) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, PI78441). Lysates were then centrifuged at 12,000 rpm for 20 min at 4°C. A total 20 µg of protein lysates was separated by 4–12% SDS-PAGE (Bio-Rad, 4561095) and transferred to a nitrocellulose membrane. The following primary antibodies were used: anti-p-eIF2α (Abcam, ab32157, 1:2000), anti-T-eIF2α (Cell Signaling, 9722s, 1:1000), anti-puromycin (Millipore, MABE343, 1:2000), anti-BIP (Cell Signaling, 3177s, 1:1000), anti-GADD34 (Proteintech, 10449–1-AP, 1:500), anti-ATF4 (Santa Cruz, sc-390063, 1:500), anti-CHOP (Thermo Fisher, MAI-250, 1:500), anti-XBP-1-spliced (Cell Signaling, 82914s, 1:1000), and anti-actin (Sigma, A2066, 1:2000). Quantification of Western blot bands were performed by Image Lab Software (Bio-Rad).

Kidney lysates were electrophoresed on SDS-PAGE gels (Invitrogen Inc., Grand Island, NY USA) and transferred to nitrocellulose membranes. These membranes were blocked in TBST containing 5% non-fat milk for 1 h (room temperature). Immunoblotting was performed with primary antibodies: anti-p-Smad2, anti-p-Smad3, anti-VEGFR2, and anti-PDGFR beta and anti-GAPDH, anti-SDHA, anti-ATPB, anti-p-PERK, anti-p-eIF2α, anti-TeIF2α (Cell Signaling Technology Inc., Danvers, MA, USA), anti-TGFRII (Santa-Cruz Biotechnology, Inc, Dallas, TX, USA), at 4°C overnight. Next, horse peroxidase-labelled appropriate secondary antibodies were added. The plotted proteins were visualized by ECL detection system (Thermo Fisher Scientific, Rockford, IL USA). Densitometric analysis was performed using ImageJ version 1.440.