Fig. 2

USP7 interacts with and stabilizes FTO A Endogenous protein interactions in HEK 293 T lysates were confirmed by immunoprecipitation with anti-USP7 or anti-FTO and immunoblotting with anti-FTO or anti-USP7. B, C Interactions between exogenous proteins were confirmed in HEK 293 T cells. Lysates from HEK 293 T cells that had been transfected with the USP7 and FTO plasmids were used for an immunoprecipitation reaction with anti-Myc/Flag. D FTO mRNA expression in HEK 293 T cells treated with shNC and shUSP7. E, F Western blotting revealed the levels of FTO and USP7 proteins in HEK 293 T cells transfected with shUSP7 or Myc-USP7 plasmids. G Immunoblotting with anti-FTO and anti-Myc was performed to analyze cell lysates after transfection of HEK 293 T with escalating doses of Myc-tagged USP7 (WT or C223S mutant). H The FTO protein levels in Myc-Vec and Myc-USP7 HEK 293 T cells were determined by immunoblotting with anti-FTO and anti-USP7 in the presence of cycloheximide (CHX, 10 g/mL) at the designated time points. I The FTO expression levels in shNC and shUSP7 HEK 293 T cells were determined by immunoblotting with FTO and USP7 antibody with CHX (10 g/mL) at the designated time points. J HEK 293 T cells that had been treated with MG132 and had USP7 knocked down before being harvested resulted in immunoprecipitated lysates that were examined using the relevant antibodies. K Lysates from transfected HEK 293 T cells with Myc-tagged USP7 (WT) or Myc-tagged USP29 (C223S), together with HA-tagged Ub and Flag-tagged FTO, were generated after immunoprecipitation with anti-Flag and immunoblotting with anti-HA and anti-Flag. L The Myc-USP7, Flag-FTO and the indicated HA-Ub, Lys0, Lys48-only, or Lys63-only plasmids were co-transfected into HEK 293 T cells before the FTO ubiquitylation linkage was assessed. M HEK 293 T cells with wild-type Ub or Lys48R mutation were cultured for 72 h in the presence of shNC or shUSP7. Anti-FTO and anti-USP7 immunoblotting was performed to evaluate the cell lysates