Jun transcript and protein levels correlate with Mef2c levels. (A) Quantitative real-time PCR was performed on M/E myeloid cells established from BM from either Mef2c^fl/− or Mef2c^+/+ mice and subsequently exposed to CRE recombinase. RNA levels were normalized against Hprt values, and the expression levels found in Mef2c^+/+ cells were set as 1. Shown is one experiment performed in duplicate. (B) To determine the effect of increased levels of Mef2c expression, Mef2c^+/+ M/E cells were infected with either Mef2c/GFP or GFP control vectors and sorted for GFP expression. Western blot analysis was performed on whole-cell lysates, and the indicated proteins were visualized using the appropriate antibodies. It should be noted that retroviral Mef2c RNA levels in the transduced M/E cells were equivalent to that observed in tissue macrophages (data not shown). (C) Although C/EBPα protein levels were reduced, no significant difference in RNA levels were detected by quantitative RT-PCR (mean of 2 quantifications). Hprt transcript levels were used to normalize transcript levels. (D) LPS stimulation increases the production of c-Jun in cells expressing Mef2c. FDC-P1 cells were engineered to express Mef2c by retroviral transduction and then subjected to LPS for 1 hour. Western blot analysis shows increased levels of c-Jun in Mef2c cultures. A vertical line has been inserted to indicate repositioned gel lanes. (E) Quantitative RT-PCR analysis demonstrates an increase in Jun transcripts after LPS treatment in Mef2c-overexpressing cells. Hprt analysis was used to normalize transcript levels; Jun transcript levels are shown relative to that found in NIH3T3 cells. Error bars denote standard deviation from the mean.