Figure 5. TRO post-translationally regulates Pdx1 and Mafa.

(A, B) After transfection with Flag or Flag-ubiquitin for 24 h, INS-1 cells were treated with GS with or without 20 µmol/l TRO for 16 h before harvesting. Ten percent of the total cell lysate was used to measure protein expression by immunoblotting (A). The rest of the total cell lysate was subjected to co-immunoprecipitation with anti-Flag, and the product was analyzed by immunoblotting (B). (C, D, E) INS-1 cells were divided into three groups (NG+DMSO, GS+DMSO and GS+20 µmol/l TRO). After the indicated treatments for 2 h, cells were co-cultured with 50 µmol/l cycloheximide for 2, 4, 8 or 12 h. Total proteins were extracted and analyzed using immunoblotting (C). The half-lives of Pdx1 (D) and Mafa (E) were calculated.