Sequestration of PPARγ by MEK1 in the nucleus of atRA treated 3T3-L1 adipocytes.
PPARγ colocalization with MEK1 in control and atRA (100 nM, 48 h) and DM containing CoA treated adipocytes were monitored by (A) Co-immunoprecipitation in the nuclear extract. (B) Adipocyte differentiation as monitored by Oil Red O staining, in response to ectopic expression of FLAG tagged wild type MEK1 and export deficient constitutively active ΔN-EE-MEK1 in endogenously silenced MEK1 background. (C) Colocalization of FLAG-MEK1 (WT and ΔN-EE-MEK1) with endogenous PPARγ, as monitored by the co-immunoprecipiation experiment in the nuclear extract is shown in the right panel. Other immunoblots, RT-PCR constitute the inputs. (D) FLAG tagged wild type MEK1 and ΔN-EE-MEK1 were ectopically transfected into 3T3-L1 cell line. qPCR was performed in these ectopically expressed cells to analyse the expression of PPARγ target genes. (E) The ChIP assays was performed on these target genes promoters to examine the recruitment of PPARγ. The results are expressed as relative to untreated cells after normalization to 18S rRNA. Error bars show SD. *P<0.05 vs. controls. Four biological repetitions of experiments, each of which was conducted in triplicate.