Mouse anti g3bp

Cells were plated on glass coverslips coated with poly-D-Lysine. 24h after induction of 97QP-GFP, stress granule assembly was stimulated by addition of 0.5 mM of sodium arsenite for 45 min at 37°C, 5% CO₂. After stress, cells were washed three times with PBS and fixed with 4% PFA in PBS for 10 min at room temperature, permeabilized with 0.5% Triton in PBS for 5 min at room temperature, washed with PBS and incubated with blocking buffer (4% BSA in PBS) for 1h at room temperature. Primary antibodies were diluted in blocking buffer and incubated for 1 h at room temperature. To monitor stress granule formation, goat anti-TIA-1 (Santa-Cruz, Dallas, TX; SC-1751; 1/100) and mouse anti-G3BP (BD Biosciences, Franklin Lakes, NJ; #611126; 1/1000) were used. After washing with 0.1% Tween in PBS, cells were incubated with IgG (H+L) secondary antibodies donkey anti-goat Alexa 647 or donkey anti-mouse Cy3 (Thermo Fisher Scientific, 1/500) for 45 min. Coverslips were mounted in Prolong Gold Antifade mounting medium (Thermo Fisher Scientific).

Cells were harvested and lysed with lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1% Triton X-100, 1 mM EDTA [pH 8.0] and 0.1% sodium dodecyl sulfate [SDS]) for 30 min on ice. The supernatants were collected via centrifugation at 12000 g at 4°C for 30 min. The protein concentration was determined using the Bradford assay method (Bio-Rad). Samples were boiled with SDS-PAGE loading buffer at 100°C for 10min and resolved via 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to nitrocellulose membranes. The membrane was blocked with 5% milk in phosphate buffered saline (PBS) with 0.1% Tween 20 (PBST) for 1h before being incubated with primary antibodies overnight and then incubated with secondary antibodies for another 1h. The primary antibodies used were as follows: mouse anti-HN (1:1000, Abcam), mouse anti-GAPDH (1:2500, Santa Cruz), rabbit anti-eIF2α (1:1000, CST), rabbit anti-phosphorylated eIF2α (1:1000, CST), rabbit anti-PKR (1:1000, Abcam), rabbit anti-phosphorylated PKR (1:1000, Abcam), mouse anti-G3BP (1:1000, BD Biosciences), mouse anti-HA tag (1:10000, Sigma), rabbit anti-IRF3 (1:1000, Abcam), and mouse anti-LMNB1 (1:1000, Applygen). HRP-conjugated goat anti-mouse immunoglobulin (IgG) (1:5000) and goat anti-rabbit IgG (1:5000) were used as secondary antibodies.