Rabbit anti g3bp1

A total of 5 × 10⁵ HeLa cells were plated on glass coverslips in a 24-well plate and fixed with 4% PFA (Sigma) in PBS for 20 min at RT, washed in PBS, and stored at 4 °C. Cells were permeabilized with 0.1% Triton-X100 (Sigma) in PBS for 7 min, then blocked with 0.5% bovine serum albumin (Fisher) in PBS for 1 h. Coverslips were then incubated with 200 μl of primary antibody solution for 1 h at RT and washed 3 times with PBS before incubation with secondary antibody solution containing 0.1 μg/ml DAPI solution (Sigma) for 1 h at RT. After three washes with PBS, coverslips were mounted to microscope slides with 7 μl Mowiol 488 (Sigma). Confocal microscopy was performed on a Ti-Eclipse A1MP Multiphoton Confocal Microscope (Nikon) using the Nikon acquisition software NIS-Elements AR. Primary antibody dilution: Rabbit anti-G3BP1 (1:600, Sigma), Goat anti-eIF3B (1:600, Santa Cruz), mouse anti-puromycin (1:5, http://dshb.biology.uiowa.edu/PMY-2A4), Rabbit anti-UBAP2L (1:600, Santa Cruz). Secondary antibodies were all purchased from Invitrogen: Chicken anti-mouse Alexa 488, donkey anti-goat Alexa 555, and goat anti-rabbit 488.

Cells were plated at 2 × 10⁵ in 6-well plates. At the indicated times, cells were lysed in 150 μl of 1× Gel Loading Buffer (New England Biolabs), sonicated and boiled 5 min at 95 °C. Cell lysates were separated by SDS-PAGE, and protein was transferred to nitrocellulose or polyvinylidene difluoride membranes. These were then probed with the following primary antibodies: rabbit anti-eIF2a (1:1,000, Cell Signalling), mouse anti-P-eIF2α (1:1,000, Cell Signalling), rabbit anti-G3BP1 (1:2,000, Sigma), rabbit anti-Caprin1 (1:1,000, Bethyl Laboratories), goat anti-eIF3B (1:2,000, Santa Cruz), mouse anti-GAPDH (1:20,000, Invitrogen), followed by incubation with the appropriate peroxidase-labeled secondary antibodies (Dako) and chemiluminescence development using the Clarity Western ECL Substrate (Bio-Rad). The results were acquired using the VILBER imaging system.