Mouse anti flag m2 hrp

The nitrocellulose membranes were blocked and antibodies were applied in 5 % milk in TBST (100 mM TRIS–HCl, pH7.5, 0.9 % NaCl, 0.1 % Tween-20). The antibodies used were rabbit anti-G3BP1 (Millipore, 07-1801), rabbit anti-SOD1 (Santa Cruz, sc-11407), mouse anti-FLAG M2-HRP (Sigma, A8592), rabbit anti-HA (Santa Cruz, sc-805), mouse anti-PABP1 (Santa Cruz, sc-32318) and goat anti-actin (Santa Cruz, sc-1616). All immunoblotting images were acquired using a BioRad ChemiDoc MP system.

For the RNase treatment assay and the final large scale purification for cryo-grid, RTS 100 E.coli HY Kit (5 PRIME) was used (transcription and translation coupled). 500 μL reaction was incubated at 30°C for 35 min with 12.6 pmol PCR-product template generated upon the ∆SS-VemP construct. RNase was added to the reaction where indicated, and incubated at 30°C for an additional 10 min. For the time course, the same amount of PCR-product template was used with the PURExpress In Vitro Protein Synthesis Kit (New England Biolabs #E6800S, transcription and translation coupled). Reactions were incubated at 37°C for various times (25/40/55/70/85/100 min). For western blotting, reaction products were separated by either home-made or commercial NuPAGE 12% Bis-Tris gels (Invitrogen, CA, USA) with 1x MOPS buffer. Proteins were blotted to nitrocellulose membrane (Carl Roth, Germany), incubated with mouse anti FLAG M2 HRP (Sigma, Germany) and visualized by ChemiDoc MP System (Bio-Rad).