FIG. 4.

KSR1 is required for C/EBPβ phosphorylation and stabilization during adipogenesis. (A) MDI-induced C/EBPβ transcription is independent of KSR1 expression. Shown is Northern blot analysis of C/EBPβ, KSR1, and β/2 M at different times after induction of differentiation in KSR1^−/− and KSR1^+/+ MEFs. (B) KSR1 enhances C/EBPβ phosphorylation during adipogenesis. Shown is Western blot analysis of whole-cell extracts prepared at different times during differentiation of KSR1^−/− and KSR1^+/+ MEFs. Lysates were probed with the indicated antibodies to detect induction and activation of C/EBPβ during adipogenesis. (C and D) Total and phosphorylated C/EBPβ expression was quantified from the Western blots described above (B) at each time point and normalized to β/2 M levels, and the resulting data were plotted as fold stimulation following induction of differentiation. Data for Thr²¹⁷ phosphorylation are shown in C, and data for Thr¹⁸⁸ phosphorylation are shown in D. KSR1^+/+, black line; KSR1^−/−, gray line; phospho-C/EBPβ, solid line; total C/EBPβ, dashed line. Data shown in each panel are the averages of three independent experiments. (E and F) Effect of KSR1 on C/EBPβ stability. Shown is Western blot analysis of whole-cell extracts prepared from KSR1^−/− and KSR1^+/+ MEFs after 4 h of induction with differentiation medium followed by 0 to 2 h of treatment with cycloheximide (CHX). Lysates were probed for C/EBPβ and β/2 M. C/EBPβ expression at each time point was quantified and normalized to β/2 M levels, and the resulting data were plotted in F. Maximal expression was set arbitrarily to 1. Values are the means of two independent experiments.