Western Blot Analysis of Brain Tissue

Frozen brain tissue was prepared for Western blot assays as previously described (Naidoo et al., 2008 (link), 2018 (link)). Briefly, brain tissue was homogenized on ice with lysis buffer containing protease inhibitors. After centrifugation, protein concentration for each sample was determined with a BCA protein assay and samples were prepared such that each contained 20µg of protein. SDS‐PAGE gels were run as previously described (Naidoo et al., 2008 (link)), and protein bands were imaged and quantified via infrared imaging on an Odyssey scanner (LiCor). For all markers, we compared n = 5–8 for each of the groups. Primary antibodies are as follows: BiP/anti‐KDEL (1:1000, Enzo Life Sciences ADI‐SPA‐827F); GADD34 (1:500, Protein Tech 10449‐1‐AP); p‐AKT (1:500, Cell Signaling 9271); and Akt (1:500, Cell Signaling 9272). Secondary antibodies are as follows: LiCor IRDye 680RD Goat anti‐Mouse (1:10,000); LiCor IRDye 800RD Goat anti‐Mouse (1:10,000); LiCor IRDye 800RD Goat anti‐Rabbit (1:10,000); and Odyssey IRDye 680 Goat anti‐Rabbit (1:10,000).