Fig. 7.

Both Lys⁴⁷ and Lys⁴⁸ of SelK are required for PPARγ-mediated ubiquitination. a Amino acid sequence of SelK is represented. The following elements of residues and domains are marked: italic letters (Lys residues), under lined letters (TM region). The construction of SelK mutants, GFP-SelK[1–55] and GFP-SelK[1–75] has been described in the Materials and methods section. b MCF7 cells were co-transfected with Flag-PPARγ and GFP-SelK or the truncation mutants of SelK for 48 h. Lysates and immunoprecipitation samples (anti-GFP and anti-IgG) were analyzed using the indicated antibodies. Cell lysates (5% input) were used as positive control. c Transfected MCF7 cells, under same conditions as (b), were treated with 2 μM MG132 for 12 h. Lysates and immunoprecipitation samples (anti-GFP) were analyzed by western blotting with the indicated antibodies. d MCF7 cells were co-transfected with Flag-PPARγ and HA-SelK or Lys-to-Arg mutants of SelK for 48 h. Whole cell lysates were analyzed by western blotting using indicated antibodies. e Transfected MCF7 cells, under same conditions as (d), were treated with 2 μM MG132 for 12 h. Lysates and immunoprecipitation samples (anti-HA) were analyzed by western blotting with the indicated antibodies. HC heavy chain, IP immunoprecipitation