Fig 1: FUS represses exon 7 of the endogenous FUS pre-mRNA and autoregulates its own protein levels.A) FUS represses exon 7 of the endogenous FUS pre-mRNA. [γ-32P] ATP labeled RT-PCR products of endogenous FUS exon 7 splicing variants in HEK293 cells, following knockdown of FUS by siRNA (siFUS). Cycloheximide (CHX) was used to inhibit NMD. The reduction of each splice variant (exon 7-included or -skipped) by siRNA relative to the corresponding mock transfection was calculated (lane 2, 3 relative to lane1; lane 5, 6 relative to lane 4). GAPDH was used as a loading control. In each sample, the reduction of the exon 7-included variant was compared with the reduction of the corresponding exon 7-skipped variant using student's t-tests. Bar graphs represent mean ± SEM (n = 3). * P≤0.05, *** P≤0.001. B) Western blot analysis of the FUS protein and two other RNA binding proteins SF2 and hnRNPA1. Actin was used for loading control. C) Expression of EGFP-FUS downregulates endogenous FUS protein. Western blot analysis of endogenous FUS protein following expression of EGFP-FUS in HEK293 cells. Both endogenous FUS and EGFP-FUS were detected using anti-FUS antibody (10F7). β-Actin was used for loading control. The endogenous FUS protein levels were quantified. Bar graphs represent mean ± SEM (n = 3). Student's t-tests were performed. Samples transfected with EGFP or EGFP-FUS were compared with the control (mock transfection). * P≤0.05.