Fig. 2

FUS-positive SGs were regulated by arginine methylation. A HEK293T cells were transfected with the HA-empty vector or HA-UBAP2L. Co-immunoprecipitation (Co-IP) was performed with an anti-HA antibody, followed by a western blot with an anti-HA and anti-PRMT1 antibody. B and C PRMT1 siRNAs were used to knock down PRMT1 expression in HEK293T cells, and an anti-PRMT1 antibody was used to perform western blot analysis of PRMT1 siRNA-treated cell lysates (B). The relative knockdown efficiency of PRMT1 siRNAs compared to that of negative control siRNA. A bar graph was used to show the relative knockdown efficiency of PRMT1 siRNAs in comparison to a negative control siRNA (C). **P < 0.01; ***P < 0.001. The values represent the mean SEM (n = 3), one-way ANOVA, and Tukey's multiple-comparison test. (D) Anti-HA antibodies were used to immunoprecipitate HEK293T cell lysates expressing HA-UBAP2L in conjunction with either negative control siRNA or PRMT1 siRNA. Following that, western blot analysis was performed using anti-ADMA, anti-HA, or anti-PRMT1 antibodies, as appropriate. The UBAP2L arginine methylation band is denoted by an asterisk. (E) Co-IP with an anti-HA antibody was performed on HEK293T cell lysates expressing FLAG-FUS WT or FLAG-FUS R521C. Following that, western blots were performed using anti-HA, anti-FLAG, anti-PRMT1, or anti-ADMA antibodies. (F) Confocal microscopy analysis was performed to demonstrate the cellular localization of FLAG-FUS, HA-UBAP2L, and eIF4G in HEK293T cells expressing either a negative control siRNA (siNC) or PRMT1 siRNA (siPRMT1) with oxidative stress-inducing SA (0.5 mM, 1 h). FLAG-FUS and UBAP2L co-localize at eIF4G positive SGs, as indicated by the arrows in each image. Scale bar: 10 μm. G A bar graph comparing the number of FUS R521C positive SGs in siPRMT1 transfected HEK293T cells expressing FLAG-FUS R521C to siNC transfected cells. Results of siNC or siPRMT1 transfected cells are shown as mean SEM (n = 15 each). ***P < 0.001, Student’s t-test