Rabbit anti p eif2a

For Western immunoblotting analysis, treated neurons were collected in 1× LDS Loading Buffer (Thermo Fisher Scientific) and boiled for 10 min. Proteins were separated by electrophoresis in 10% NuPAGE gels (Thermo Fisher Scientific). To better detect ATF4, the gels were run a longer time (80–100 min) to separate ATF4 from a closely migrating nonspecific band. The following primary antibodies were used: rabbit monoclonal anti-ATF4 (1:1000), rabbit anti-eIF2a (1:1000), rabbit anti-p-eIF2a (1:1000), anti-CREB (1:1000), and anti-pCREB (1:2000), all from Cell Signaling Technology; and mouse anti-GAPDH (1:2000) from Imgenex. For secondary antibody, HRP-conjugated anti-rabbit and anti-mouse secondary antibody (1:5000; Thermo Fisher Scientific) was used. The ATF4 antibodies were validated by assessment in cultures in which ATF4 had been knocked down with lentiviral-delivered short hairpin ATF4 (shATF4; Corona et al., 2018 (link)).

Cells were lysed using 2X SDS-PAGE sample buffer (24 mM Tris-HCl, pH 6.8, 10% glycerol, 0.8% SDS, and 286 mM β-mercaptoethanol). Samples were subjected to SDS-PAGE on 4–20% Mini-PROTEAM^R TGX^™ gels (Bio-Rad, 4561096) and transferred to a nitrocellulose membrane (Bio-Rad, 1704159). The primary antibodies used in this study were: rabbit anti-ATF4 (Cell Signaling Technologeis, 11815s, 1:625), rabbit anti-β-actin (Cell Signaling Technologies, 4970S, 1:2500), rabbit anti-P-EIF2a (Cell Signaling Technologies, 3398S, 1:1000), mouse anti-EIF2α (Fisher Scientific, AHO0802, 1:1000), rabbit anti-β-Tubulin (Thermo Fisher, 32–2600, 1:2500), rabbit anti-UCP1 (Abcam, ab10983, 1:2500), rabbit anti-Flag (Cell Signaling Technologies, 2368S, 1:5000), rabbit anti-PKM2 (Cell Signaling Technologies, 4053S, 1:1000), rabbit anti-Tom20 (Cell Signaling Technologies, 42406S, 1:2500). Blots were incubated with antirabbit IgG (Cell Signaling Technologies, 7076S, 1:10000) or anti-mouse IgG (Cell Signaling Technologies, 7074S, 1:10000) and imaged using an analyticjena imaging system. Digital images were processed and quantified using ImageJ software.