Rabbit anti g3bp1

Cells expressing SOD1(A4V)‐GFP were exposed to heat stress for 2 h and fixed with 4% PFA (diluted in PBS) for 10 min. This was followed by washing with PBS and then permeabilization with 0.2% Triton X‐100 (diluted in PBS) for 10 min. The cells were then washed with 2× SSC. Hybridization was then performed by incubating the cells for 2 h at 37°C in 4× SSC containing 10% formamide, 5% dextran sulfate, 1% BSA, 0.5 mM EDTA, and 100 nM biotinylated oligo‐dT probe (23‐mer). This was followed with washing in 2× SSC (3 × 10 min) and blocking in 3% BSA (diluted in 4× SSC) for 1 h at room temperature. The cells were then incubated with primary antibodies (goat anti‐biotin, Sigma‐Aldrich, B3640; rabbit anti‐G3BP1, Thermo Fisher Scientific, PA5‐29455) diluted in 1% BSA (in 4× SSC) for 1 h. The cells were then washed in 4× SSC and incubated with secondary antibodies conjugated with Alexa Fluor fluorophores (Invitrogen) diluted in 1% BSA (in 4× SSC) for 1 h. The cells were then washed in 4× SSC, then washed in 2× SSC, and finally mounted in DAPI‐Fluoromount G (SouthernBiotech). The cells were then imaged using the DeltaVision imaging system (Applied Precision) as described above.