Mouse anti eif2α l57a5

Western blotting was performed as described previously^{32 (link)}. Parasite pellets were isolated using cold 0.04% Saponin (Sigma) in 1X PBS for 10 minutes as described previously^{32 (link),36 (link)}. Antibodies used for this study were: mouse anti-GFP JL-8 (Clontech, 1:3000), rabbit anti-PfEF1α (from D. Goldberg, 1:2,000), mouse anti-plasmepsin V (from D. Goldberg, 1:400), rabbit anti-PfBiP MRA-1246 (BEI resources, 1:500), rabbit anti-GFP A-6455 (Invitrogen, 1:2,000), mouse anti-eIF2α L57A5 (Cell Signaling, 1:1,000), rabbit anti-Phospho-eIF2α 119A11 (Cell Signaling, 1:1,000), rat anti-HA (Roche 3F10, 1:3000), mouse anti-Ty1 (Sigma Clone BB2, 1:1000), and mouse anti-Ub P4D1 (Santa Cruz Biotechnology, 1:1,000). Secondary antibodies used were IRDye 680CW goat anti-rabbit IgG and IRDye 800CW goat anti-mouse IgG (LICOR Biosciences, 1:20,000). The western blots were imaged using the Odyssey infrared imaging system. Polyacrylamide gels used in this study were either prepared using 10% EZ-Run protein gel solution (Fisher) or precast gradient gels (4–20%, from Biorad). Any quantification performed on western blots was done using ImageJ software. The quantification data were analyzed using Prism (GraphPad Software, Inc.).

Cells were lysed for 30 min on ice in RIPA Lysis Buffer supplemented with phosphatase and protease inhibitor cocktails (Santa Cruz Biotechnology). Lysates were cleared by centrifugation at 17000g for 10 min at 4°C. Supernatants were removed and assayed for protein concentration using the Pierce BCA Protein Assay Kit (Thermo Fisher). Western blotting was performed using Novex system (Invitrogen). Equal amounts of proteins were subjected to SDS-PAGE and transferred to PVDF membranes (Thermo Fisher). Membranes were incubated with primary antibodies at 4°C overnight. Membranes were washed in TBS-Tween and then incubated with HRP conjugated anti-mouse or anti-rabbit secondary antibodies (Jackson Immunoresearch) for 1 hr at room temp and developed using SuperSignal^™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher). Signal detection was captured using Odyssey Fc system (LI-COR; Figure S6). The following primary antibodies were used: mouse anti-eIF2B5 (B-7, 1:50; Santa Cruz Biotechnology); mouse anti-β-Actin (2D4H5, 1:3000 for WB; Proteintech); mouse anti-Puromycin (12D10, 1:1000; Millipore); mouse anti-EIF2α (L57A5, 1:500; Cell Signaling); rabbit anti-phospho-EIF2α Ser51 (119A11, 1:500; Cell Signaling); rabbit anti-FBXO32 (EPR9148(2), 1:500; Abcam); mouse anti-V5 (V5-10, 1:3000 for WB; Sigma-Aldrich).