Figure 3.

The binding activity of Q14 peptide with USP30. (A) Increasing concentrations of USP30 were incubated with FITC-labeled peptide FITC-Q14 and detected by fluorescence polarization. Data are represented as mean ± s.d. of two independent experiments. (B) A172 cell lysates were incubated with biotin-labeled peptide B-Q14, biotin-labeled peptide-Qscr, biotin and DMSO at 4°C. SA beads were added into the mixture and used for Western blot analysis through an anti-USP30 antibody. GAPDH was used as a loading control. (C) Representative immunoblots for cell lysate based thermal shift assay carried out in A172 cells treated with DMSO or Q14 peptide (50 µM). Western blots were developed using an anti-USP30 antibody. GAPDH was shown as a loading control. (D) Graphical summary of the results of a cell lysate based thermal shift assay of A172 cells treated with DMSO or Q14 peptide (50 µM). Data are presented as mean ± s.e.m. from three independent experiments. Full-length gels for B and C are in Fig. S8.