FIGURE 3.

Ubiquitin‐specific protease 28 (USP28) interacts with and modulates ubiquitination of transcription factor 7‐like 2 (TCF7L2). (A) Western blot analysis of TCF7L2 in HEK293T cells treated with MG132 (20 μM) for 3 h followed USP28 depletion by shRNA‐mediated knockdown. (B) Half‐life analysis of TCF7L2 protein in HEK293T cells with USP28 depletion. Cells were treated with cycloheximide (CHX, 100 μg/ml) for the indicated time periods and harvested for analysis. Protein band intensities were measured with Image J software and plotted. (C) Immunoprecipitation of S·tag‐Flag‐TCF7L2 expressed in HEK293T cells followed by western blot analysis. (D) Immunoprecipitation of S·tag‐Flag‐USP28 expressed in HEK293T cells followed by western blot analysis. (E) Immunoprecipitation of S•tag‐Flag‐USP28 (full length and truncation mutants, see F) in HEK293T cells to examine the interaction between USP28 and TCF7L2. (F) Schematic diagram of the deletion mutants of USP28. (G, H) Ubiquitination assay of TCF7L2 in HEK293T cells with USP28 depleted (G) or overexpressed (H). Cells were treated with MG132 (10 μM) for 8 h before sample collection. IP, immunoprecipitant