Figure 2.

USP29 deubiquitinates and stabilizes TWIST1. A) MDA‐MB‐231 cells stably expressing control (Ctrl) or USP29 shRNAs (#1 and #2) were generated and western blot was performed with indicated antibodies. B) Total RNA was isolated from cells in (A). The expression of TWIST1 mRNA in cells was determined by quantitative PCR. Transcript levels were determined relative to GAPDH mRNA level and normalized relative to control. The results represent mean ± s.d. from three independent experiments. C) MDA‐MB‐231 cells stably expressing control or USP29 shRNAs were treated with vehicle or MG132 (10 µm) and western blot was performed with indicated antibodies. D) Cycloheximide pulse‐chase assay was performed in cells as in (A); E) the relative level of TWIST1 to β‐actin was measured by image J. The results represent mean ± s.d. from three independent experiments. F) Cells were cotransfected with indicated plasmids and Ni‐NTA bead was used to pull down His‐tagged ubiquitin, and the polyubiquitylated TWIST1 protein was examined by western blot. G) Cells were transfected with HA‐TWIST1 and treated with MG132 (10 µm) for 10 h. Cell lysates were immunoprecipitated with anti‐HA antibody and incubated with purified GST, GST‐USP29, or GST‐USP29 C294S mutant in a cell‐free condition. The polyubiquitylated TWIST1 protein was detected by anti‐ubiquitin antibody. H) MDA‐MB‐231 cells stably expressing control or USP29 shRNAs were transfected with indicated plasmids and treated with MG‐132 (10 µm) for 10 h. Cell lysates were subjected to immunoprecipitation with anti‐Flag antibody and the ubiquitination of TWIST1 protein was examined by western blot.