Gadd34

Human macrophage colony-stimulating factor (M-CSF) and RANKL was purchased from Peprotech (Rocky Hill, NJ, United States). Rapamycin was obtained from Calbiochem. Antibodies were used for immunoblotting as follows: NFATc1 (Santa Cruz Biotechnology; sc-7294); α-tubulin (Sigma-Aldrich; T9026); phosphorylated p70S6K, p70S6K (Cell Signaling Technology; 9234, 2708); and GADD34 (Proteintech; 10449-1-AP). A mouse CTX-I and P1NP ELISA kits were purchased from Cloud-Clone (Wuhan, China).

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).

Cells were lysed in buffer H (10 mM HEPES, pH 7.9, 50 mM NaCl, 500 mM sucrose, 0.1 mM EDTA, 0.5% (v/v) Triton X-100, 1 mM PMSF, 1X Complete protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany)) supplemented with phosphatase inhibitors (10 mM tetrasodium pyrophosphate, 17.5 mM β-glycerophosphate, and 100 mM NaF [25 (link), 27 (link)]) for detection by antibodies directed against phospho-eIF2α (Cell Signaling Technology, Danvers, MA, USA), eIF2α (gift from Prof. Dr. D. Ron), KDEL (Enzo Life Sciences), GADD34 (ProteinTech, Chicago, IL, USA), puromycin (Millipore, Billerica, MA, USA), β-actin and GAPDH (CellSignalling), or in sample buffer (0.2 M Tris-HCl pH 6.8, 16% [v/v] glycerol, 4% [w/v] SDS, 4% [v/v] 2-mercaptoethanol, 0.003% [w/v] bromophenol blue) for detection by antibodies directed against (phospho-) p38 MAPK (both CellSignalling). The proteins in the samples were separated using a 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane. After blocking with PBS containing 0.05% Tween-20 (v/v) and 5% skimmed-milk (w/v), the membrane was incubated overnight with the primary antibody (1:1000) in TBS with 0.05% Tween-20 (v/v) and 5% BSA (w/v) at 4°C. Next, the membrane was incubated with HRP-labelled anti-mouse or anti-rabbit antibody (Sigma) in blocking buffer for 1 hour and developed using ECL (ThermoScientific).