Figure 3.

p38 regulates transcription factor recruitment. (A) MDER fibroblasts were either untreated (0 h) or infected with MKK6E retrovirus (+) or empty vector control retrovirus (-) and induced to differentiate for 12, 24, or 48 h. ChIP was performed with broad-specificity Mef2 antiserum, and multiplex PCR for the indicated promoters was performed with pancreatic amylase (Amy) as an internal control. Input chromatin was amplified over a 30-fold range to verify linearity of the assay. Graphs indicate IP promoter fold enrichment relative to input chromatin. Data for a representative ChIP are shown with error bars indicating S.E.M. for triplicate PCR. (B) Mef2 ChIP demonstrating that active MyoD is necessary for Mef2 binding, and that the broad-specificity Mef2 antisera identifies Mef2 binding both with and without exogenous Mef2D expression. (NS) Nonspecific IgG control ChIP; (E₂) β-estradiol to induce MyoD activity. (C) ChIP using Mef2D monoclonal antibody shows Mef2D is not bound at early times in the absence of exogenous Mef2D. (GFP) A control retrovirus; (K6 + 2D or 6 + D) a combination of the MKK6E and Mef2D retroviruses. (D) ChIP using MyoD antisera shows p38 regulation of MyoD binding but no dependence on Mef2D. Input titration shown in C. (Empty) Control virus without effector gene.