G3bp1

The following antibodies were used in this study: ASC (#67824, CST), α‐tubulin (#RM2007, Ray antibody Biotech), β‐actin (#PM053, MBL), Caspase‐1 (p20) (AG‐20B‐0042, AdipoGen), DDX3X (ab271002, Abcam), eIF2α (#5324, CST), Phospho‐eIF2α (Ser51) (#3398, CST), FAM69C (home‐made), FLAG (#F3165, Sigma‐Aldrich), G3BP1 (sc‐365338, Santacruz), GAPDH (#RM2002, Ray antibody Biotech), GFAP (#12389, CST), GFP (#RM1008, Ray antibody Biotech), GM130 (#2296, CST), HA (#H3663, Sigma‐Aldrich), HRI (sc‐365239, Santacruz), Iba1 (019–19741, Wako), PERK (#5683, CST), Puromycin (MABE341, Millipore).

Cells were washed twice with ice-cold PBS, then lysed in SDS lysis buffer containing 1× protease inhibitor cocktail (Roche Applied Science, Germany). The total protein concentration in the cell lysate solution was then determined via the BCA protein assay (Thermo Fisher Scientific, USA). Protein samples (40 μg) were separated by electrophoresis on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, USA). After being blocked with 5% skim milk (BD Biosciences, USA) for 1 h, the membranes were incubated with specific primary antibodies; β-actin (Affinity Biosciences, USA, T0022; 1:4000), GAPDH (Immunoway Biotechnology, USA, YM3040; 1:4000), YBX1 (Santa Cruz Biotechnology, USA, sc-101,198; 1:1000), G3BP1 (Santa Cruz, sc-98,561; 1:1000), SPP1 (Abcam, USA, ab-69,498; 1:2000), p-p65 (Cell Signaling Technology, USA, #3033; 1:1000), and p65 (Cell Signaling Technology, USA, #8242; 1:1000), overnight at 4 °C. After washing with TBST, the membranes were incubated with HRP-conjugated anti-mouse (Affinity, S0002) or anti-rabbit (Affinity, S0001) secondary antibodies for 1 h and visualized with ECL system (Millipore, USA).

For images in Figs. 2 and 5, iPSC-CMs were fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 20 min and then extracted for 5 min with 0.3% Triton X-100 and 4% PFA in PBS as previously described (Burnette et al., 2014). Cells were washed three times in 1 × PBS. Cells were blocked in 5% BSA in PBS for at least 30 min. Primary antibodies were diluted in 5% BSA + 0.3% Triton X-100. RBM20 antibody (ThermoFisher, Cat#PA5-58068) was used at 1:500, DDX6 (MilliporeSigma, Cat# SAB4200837), and G3BP1 (SantaCruz Biotechnology, Cat# sc-365338) antibodies were used at 1:200. For actin visualization, phalloidin-488 (ThermoFisher Scientific, Cat# A12379) in 1x PBS (15 μl of stock phalloidin per 200 μl of PBS) was used for 3 h at room temperature. DNA was visualized via DAPI staining for 30 min at room temperature (final concentration 1.2 μM). Cells were kept and imaged in 1× PBS.