FIGURE 2.

USP11 negatively regulates KLF4 through K63‐linked ubiquitination. A, Flag‐KLF4 and HA‐Ubiquitin (WT‐ or KO‐Ub) plasmids were cotransfected with empty vector or Myc‐USP11 into HEK293 cells. KLF4 ubiquitination was determined by IP of KLF4 with anti‐Flag Abs beads and immunoblotting with anti‐HA antibody. B, Flag‐KLF4 and HA‐Ub plasmids were cotransfected with USP11 (WT or its active‐site mutant C318S). KLF4 protein in lysates of transfected cells was subjected to IP with anti‐Flag Abs beads and KLF4 ubiquitination was analysed by anti‐HA antibody. C, Ubiquitinated Flag‐KLF4 proteins in transient transfected HEK293 cells were pulled down by anti‐Flag Abs beads, followed by incubation with purified GST‐USP11 proteins. KLF4 ubiquitination levels in vitro were determined by Western blotting with anti‐HA antibody. D, KLF4 was cotransfected with HA‐ubiquitin (WT, K48 or K63) and USP11 into HEK293 cells. KLF4 was immunoprecipitated using anti‐Flag Abs beads and immunoblotting with anti‐HA antibody. E, Endogenous KLF4 was isolated using anti‐KLF4 antibody after cotransfection Myc‐USP11 and HA‐K63 Ub plasmids into HepG2 cells and immunoblotting with anti‐HA antibody