Anti g3bp1

Mouse anti-p24 was obtained from the National Institutes of Health AIDS Reference and Reagent Program. Anti-eIF4A (provided by Dr Simon Morley, University of Sussex, UK); anti-G3BP1 (provided by Dr Imed Gallouzi, McGill University, Canada); anti-TIAR (Santa Cruz Biotechnology); anti- eIF2α-P (Abcam); anti-eIF4GI (provided by Dr Nahum Sonenberg, McGill University, Canada); anti-eIF3b (Santa Cruz Biotechnology) and AlexaFluor fluorophore conjugates (Life Technologies).

Proteins were resolved through SDS-PAGE using 4–15% precast Mini-PROTEAN TGX Gels (Bio-Rad) and NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific) before transferring to a PVDF membrane (Bio-Rad). Immunodetection was achieved with 1:5,000 anti-ZAP (Abcam, Cambridge, United Kingdom); 1:5,000 anti-TRIM25 (BD Biosciences), 1:1,000 anti-HA (Roche Life Science), 1:5000 anti-V5 (Invitrogen), 1:2,500 anti-myc (Cell Signaling Technology, Danvers, MA), 1:20,000 anti-FLAG (Sigma-Aldrich), 1:500 anti-G3BP1 (Santa Cruz, Dallas, TX), 1:1,000 anti-G3BP2 (Assay Biotech, Fremont, CA), 1:1,000 anti-UPF1 (Cell Signaling Technology), 1:10,000 anti-NME1 (Abcam), 1:2,000 anti-PABPC4 (Proteintech, Rosemont, IL), and 1:20,000 anti-actin-HRP (Sigma-Aldrich). Primary antibodies were detected with 1:20,000 goat anti-mouse HRP (Jackson ImmunoResearch, West Grove, PA), 1:20,000 goat anti-rabbit HRP (Thermo Fisher Scientific), or 1:20,000 donkey anti-rat HRP (Jackson ImmunoResearch). Proteins were resolved on a 4–15% Mini-PROTEAN TGX gel (Bio-Rad, Hercules, CA) and visualized using ProSignal Pico ECL Reagent (Genesee Scientific, San Diego, CA) on a ChemiDoc (Bio-Rad). Quantification of western blots was performed using ImageJ.

Cells were grown to ∼75% confluency and exposed to stress conditions, then fixed with 3.7% formaldehyde, permeabilized using 0.2% Triton X-100 in PBS, and then blocked using 100% FBS. Cells were then stained with primary antibodies incubated for 2 h at room temperature or overnight at 4 °C, washed, and treated with a fluorescent secondary antibody for 1 h. Some of the primary antibodies that were used were anti-PABPC1 (Santa Cruz, sc-32318), antiG3BP1 (Santa Cruz, sc-81940), and anti-LSM14A (ABclonal Cat No: A16682). After another wash, the 35-mm MatTek glass bottom culture dishes were imaged on the ISS Alba FLIM 2-photon confocal microscope using a 100X/1.49 oil TIRF objective to microscopically count the number of particles formed under different conditions per μm². For each condition, 10 to 12 cells were randomly selected, and z-stack measurements were taken (1.0 μ/frame). Analysis was performed using ImageJ where each measurement was thresholded before averaging the number of particles per frame per measurement. Statistical significance was calculated using SigmaPlot with a Student’s t test, Tukey test, or ANOVA on Ranks.