Detailed Western Blotting Protocol

Western blotting was performed largely as described previously^{13 (link)}. Protein lysates were prepared by lysing astrocytes with boiling 1× Laemmli buffer (Boston BioProducts, #BP-111R) followed by boiling at 95 °C for 5 min. SDS–PAGE was performed using Bolt 4–12% Bis-Tris Plus gradient gels (Invitrogen, #NW04125BOX). Western blotting was performed by transferring proteins onto a PVDF membrane (Millipore, #IPVH15150) in 1× NuPAGE buffer (Thermo Fisher, #NP00061). Membranes were blocked in 10% milk (Laboratory Scientific, #M0841) in TBS-T (Boston BioProducts, #IBB-180–2L). Primary antibodies used in this study were: rabbit anti-MAFG (Genetex, #GTX114541, 1:1,000), rabbit anti-GAPDH (Cell Signaling Technology, #2118S, 1:1,000), rabbit anti-cyclophilin B (Thermo Fisher Scientific, #PA1027A, 1:1,000), and rabbit anti-MAT2α (Novus Biologicals, #NB110–94158, 1:1,000). The secondary antibody used in this study was anti-rabbit IgG-HRP conjugate (Cell Signaling Technology, #7074S, 1:1,000). HRP-conjugated blots were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, #34095) and CL-XPosure Film (Thermo Fisher Scientific, #34090). Some HRP-conjugated blots were developed using the KwikQuant imaging system (Kindle Biosciences). Film was developed using a M35A X-OMAT Film Processor (Kodak).