USP29 interacts with TWIST1. A) List of TWIST1‐associated proteins identified by mass spectrometric analysis. MDA‐MB‐231 cells stably expressing Flag‐TWIST1 were generated and TWIST1 complexes were subjected to mass spectrometric analysis. B,C) MDA‐MB‐231 cell lysates were subjected to immunoprecipitation with B) IgG, anti‐TWIST1 or C) anti‐USP29 antibodies. The immunoprecipitates were blotted with indicated antibodies. D) Purified recombinant GST, GST‐USP29 and His‐TWIST1 were incubated in vitro and the interaction between USP29 and TWIST1 was examined. CBS, Coomassie blue staining. E) Schematic showing the generation of the USP29 (full‐length (FL), M1 (1‐286aa), M2 (287‐486aa), M3 (287‐686aa) and M4 (687‐922aa)) constructs. F) Full length Flag‐USP29 or its truncation mutants were co‐expressed with HA‐TWIST1 in 293T cells. Cell lysates were subjected to immunoprecipitation with S‐protein Agaroses. The immunoprecipitates were blotted with indicated antibodies. G) Co‐localization of endogenous USP29 and TWIST1 in MDA‐MB231 cells was detected by immunofluorescence (IF) staining. Scale bars, 25 µm.