Mef2c/d proteins are phosphorylated after pre-BCR signaling. (A) Western blot analysis of Blnk-ERt2, phospho-Blnk-ERt2, phospho-Erk1/2, and Erk1/2 in Blnk-deficient pre-B cells transduced with Blnk-ERt2 and stimulated for 3 minutes with an IgH antibody (µHC) and tamoxifen (4-OHT) as indicated. Protein molecular weights are indicated in kilodaltons. (B) Relative transcript levels of Mef2c and Mef2d in pre-B cells stimulated for the indicated time periods identified by RNA-seq analysis and confirmed by RT-PCR. Error bars represent ± SD. **P < .01; ***P < .001; ns, not significant. (C) Immunoblot analysis and quantification of Mef2c and Mef2d proteins in Blnk-ERt2–overexpressing pre-B cells stimulated as indicated. Quantification was performed using the Odyssey Image scanner system (LI-COR Biosciences). Numbers indicate the level of Mef2c/d protein relative to that in the untreated control (first lane). Gapdh served as a loading control. (D) Western blot analysis of phospho-p38, phospho-Erk5, and Erk5 in Blnk-deficient pre-B cells as described in panel A. (E) Immunoprecipitation of Mef2c/d in Blnk-ERt2–overexpressing pre-B cells stimulated with IgH and 4-OHT for 10 minutes and with or without treatment with Mek inhibitor U0126. Phosphorylation of the Mef2 proteins at the indicated serines was visualized using specific antibodies. Gapdh served as a loading control. Twenty percent of the cell lysate was used as input control. Results shown are representative of at least 2 independent experiments performed. Protein molecular weights are indicated in kilodaltons.