Fig. 3.
USP7 regulates misfolded mutant SOD1 protein levels through the transcription factor SMAD2. (A) The SMAD transcriptional activity was increased in HEK293 cells upon USP7 knockdown (Left) or inhibition of USP7 by the small-molecule drug HBX41108 at 2 μM (Right), as measured by the SMAD response element (SMAD-RE)–mediated luciferase activity assay (n = 3; *P < 0.05, ****P < 0.0001). (B) The protein levels of SMAD2, but not those of SMAD3, were increased upon USP7 knockdown (n = 3; **P < 0.01). (C) Endogenous SMAD2 was coimmunoprecipitated when Flag-USP7 expressed in HEK293 cell was pulled down by the anti-Flag antibody but not the IgG control. (D) Immunoblot analysis of SOD1G85R in cells with USP7 or SMAD2 knockdown, or double knockdown, indicated that loss of SMAD2 abolished the effect of USP7 on the regulation of SOD1G85R levels in both the supernatant and pellet fractions (n = 4; *P < 0.05, **P < 0.01). Error bars indicate ± SEM.