Figure 2
Propagation of misfolded HuWtSOD1 depends on endogenous HuWtSOD1 substrate.
Representative immunoblots of quantitative immunoprecipitation of SOD1 proteins from untransfected (a) or SOD1-siRNA transfected (d) HEK293 cells incubated for 20 h with conditioned media from TDP-43 or FUS-transfected HEK293 cells. Immunoprecipitation studies were performed using a rabbit polyclonal pan-SOD1 antibody, SOD100 and misfolded SOD1-specific mouse monoclonal antibodies, 3H1 and 10C12. Mouse IgG2a isotype control was used as negative control and blots were probed with pan-SOD1 antibody. Lysate pull-down signals from 3H1 (b) and 10C12 (c) were normalized to total immunoprecipitable SOD1 in each lysate and expressed as a percentage of total SOD1. We used non parametric one-way ANOVA to established statistical significance between cells incubated with conditioned media from FUSR495x, FUSP525L, wtTDP-43 and TDP-43ΔNLS transfected cells as compared to cells incubated with conditioned media from empty vector transfected cells. Two tailed Student’s t-test was used to demonstrate statistically significant reduction in detectable misfolded SOD1 between SOD1-siRNA treated (e, f) and the corresponding untreated (b, c) recipient cell cultures incubated with the same conditioned media. Inset in (d) confirms downregulation of SOD1 using an immunoblot probed with pan-SOD1 and actin (load control) antibodies.*,p < 0.05 ; **,p < 0.01; ****,p < 0.0001. Number of biological repeats was 9 for empty vector and wtFUS and 16 for the other constructs.
