Knockdown of Med1 or Med12 impairs CSR in CH12 cells. (A) Knockdown efficiencies were determined by Western blot on transduced cells stimulated for 48 h and sorted for GFP expression. Western blots for β-actin, Med1, Med12, and AID are shown. Theoretical molecular masses in kilodaltons are indicated. Data are representative of three experiments. (B) IgA surface expression as determined by flow cytometry in stimulated CH12 cells transduced with a lentivirus expressing a GFP reporter and shRNAs specific for AID, Med1, Med12, and a nontarget shRNA negative control. Representative plots (gated on live cells) are shown. Percentage of cells in each quadrant is indicated, and the percentage of IgA⁺ cells among the GFP⁺ population is indicated in the upper right quadrants. (C) Percentage (+SD) of CSR relative to the nontarget shRNA control from four independent experiments by gating on cells expressing GFP. CSR in cells expressing the nontarget shRNA control was set to 100%. The difference in CSR efficiency between nontarget and shRNA knockdown (Δ) is indicated below. Statistical significance versus the nontarget shRNA control (two-tailed Student's t test) is indicated. (D and E) RT-qPCR for Iμ-Cμ (D) and Iα-Cα (E) germline transcripts in transduced cells stimulated for 48 h and sorted for GFP expression. Transcript cycle threshold values were normalized to hypoxanthine-guanine phosphoribosyltransferase mRNA abundance and are presented relative to the nontarget shRNA negative control (set as 1). Statistical significance versus the nontarget shRNA control (two-tailed Student's t test) is indicated. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.0001. Data are representative of three independent experiments.