Activation of Mef2c/d target gene expression during pre-BCR signaling. (A) Activated and repressed gene expression (≥2-fold; identified by RNA-seq analysis) in Blnk-ERt2–expressing pre-B cells stimulated with IgH antibody and tamoxifen (4-OHT) for indicated time periods. (B) Relative transcript levels of a selection of genes (Egr1/2, Klf2, Zfp36, Jun, Fos, Irf4) in Blnk-ERt2⁺ pre-B cells stimulated for different time points using IgH and 4-OHT. qRT-PCR was used to confirm transcript level of a subset of genes, indicated by a star (★). (C) Immunoblot analysis of Klf2, Jun, and Irf4 in Blnk-ERt2–overexpressing cells stimulated for 6 and 24 hours with IgH and 4-OHT. Gapdh served as a control. (D) Relative expression of Klf2 and Jun in Blnk-ERt2⁺ pre-B cells stimulated with IgH and 4-OHT for 30 minutes and treated with different concentrations of Mek inhibitor U0126 as indicated. (E) Analysis of the impact of Mef2c and Mef2d mutants on Klf2 expression using a reporter gene assay. Cos-7 cells were transfected with a luciferase expression plasmid containing a Klf2 promotor with a Mef2c/d wild-type expression plasmid or Mef2c (S59A, T293A, T300A, S387A) or Mef2d (S180A) phosphorylation mutants or empty control plasmid. Firefly luciferase activity (relative to renilla luciferase) was determined. **P < .01; ***P < .001; ns, not significant. Results shown are representative of 2 independent experiments performed.