Figure 4.

RB regulates half-life and stability of Pdx-1 protein. (A) Treatment of MIN6 cells with RB siRNA (+), but not control siRNA (−), results in reduced level of RB and Pdx-1 protein. Tubulin levels serve as control. (B) Infection of MIN6 or wild-type mouse islets with RB shRNA lentiviruses, but not control scrambled lentiviruses, results in reduced RB and Pdx-1 protein levels. (C) Wild-type islets were infected with control (−shRB) or RB shRNA (+shRB) for 72 h followed by no treatment (Cont), or incubation for 7 h with DMSO or cycloheximide (CHX). Islet protein lysates were immunoblotted with Pdx-1 and tubulin antibodies. (D) Wild-type islets were incubated with DMSO (−) or Cdk4 inhibitor (+). Islet protein lysates were immunoblotted with Pdx-1 and tubulin antibodies. (E) Cdk4^WT or Cdk4^R24C islets were incubated for the indicated hours with cycloheximide (CHX). Islet protein lysates were immunoblotted with Pdx-1 and tubulin antibodies. (F) Empty vector (Mock), wild-type Pdx-1 or the RIM mutant Pdx-1-M was transfected in Rb-deficient Saos-2 cells along with vector alone pcDNA3 or HA-RB followed by immunoblot with anti-c-myc, anti-HA and tubulin antibodies. (G) Rb-deficient Saos-2 cells were transfected with empty vector (Mock), wild-type Pdx-1 or the RIM mutant Pdx-1-M and Flag-tagged ubiquitin without (lanes 2–4) or with (lanes 5–7) HA-RB. Ubiquitinated Pdx-1 was assessed by immunoprecipitation with anti-Pdx-1 antibody followed by immunoblot with anti-Flag (to detect ubiquitinated Pdx-1) and anti-Pdx-1 antibodies (lower panel; arrow points to Pdx-1 under the IgG band) to detect levels of Pdx-1.