Mouse anti gfp jl 8

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.).

For Western blot analysis, cells were lysed in cold BC100 or FLAG buffer (50 mM Tris-HCl, pH 7.9, 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1% Triton X-100, 0.2% Sarkosyl, 10% glycerol, and freshly supplemented protease inhibitor cocktail) and then total protein concentrations were determined by Bradford method using the Bio-Rad protein assay kit. The cell extracts were boiled in SDS loading buffer, and then equally loaded and separated in polyacrylamide gels. Proteins were then transferred to Hybond ECL membrane (GE healthcare) and incubated overnight with primary antibodies. Rabbit polyclonal Mdmx antibody (A300-287A) was purchased from Bethyl Laboratories. Mouse monoclonal antibody for Peli1 (F-7), p53 (DO-1), Mdm2 (SMP14) and Myc (9E10) and rabbit polyclonal antibodies for p21 (C19) and Puma (H-136) were purchased from Santa Cruz Biotechnology. Rat anti-HA monoclonal antibody (3F10) was purchased from Roche. Mouse anti-GFP (JL-8) and β-actin (AC-15) monoclonal antibodies were purchased from Clontech and Sigma, respectively. HRP-conjugated secondary antibodies were purchased from GE Healthcare. Western blot signals were detected on auto-radiographic films after incubating with ECL (GE healthcare) or Super Signal West Dura reagents (Thermo scientific).