Fig. 2.

Ectopic expression of Prep1 destabilizes Meis1a in Prep1^i/i MEFs. (A) Overexpression of Prep1 reduces the level of Meis1a in Meis1a-overexpressing Prep1^i/i cells. Anti-FLAG antibody was used to check FLAG-Meis1a and FLAG-Prep1 expression in nuclear lysates of cells infected with Meis1a or Meis1a-Prep1. Anti-Meis1–specific antibody was used to check total Meis1a level in these cells. Proliferating cell nuclear antigen (PCNA) was used as a protein loading control. Bar graph shows densitometric analysis of Meis1a values normalized to the levels of PCNA. The data shown derive from a single experiment but are representative of at least three experiments showing the same result. (B) Effect of Prep1 overexpression on Meis1 mRNA level. Bar graph represents Meis1 transcript level measured by real-time PCR in Meis1a and Meis1a-Prep1–overexpressing Prep1^i/i MEFs. The data shown derive from three independent triplicate experiments. (C) Meis1a degradation is proteasome-dependent in the presence of reexpressed Prep1. Exponentially growing passage-35 Prep1^i/i MEFs transduced with both Meis1a and Prep1 were treated with 20 μM MG132 for the indicated time windows. Total extracts were processed for immunoblotting using anti-Meis1 antibody. Vinculin was used as a protein loading control. The graph represents the percentage of Meis1a accumulation on proteasome inhibition. (D) Prep1 overexpression decreases Meis1a stability. Passage-35 MEFs were treated with 10 μg/mL CHX and collected at the indicated time points. A specific Meis1 antibody was used for immunoblotting. The graph shows the percentage of residual Meis1a at various times after the addition of CHX. The levels at t = 0 are considered 100%. The data represent the average of three experiments. Error bar indicate SD (*P < 0.05).