Regulation of IRF4 by C/EBPβ. (A) The human WT-C/EBPβ (also known as NF-IL6) cDNA is depicted as well as the region (an Spl1 fragment encoding amino acids 41-205) deleted to generate the DN-C/EBβ cDNA. These cDNAs inserted into empty vector (EV) pCDNA3.1 were used for the transfection studies. Transfection of the WT-C/EBPβ cDNA in MM cells results in overexpression. MM cell lines, MM.1S, H929, and RPMI-8266, were transfected with the EV, (B) DN-C/EBPβ, or (C) WT-C/EBPβ followed by G418 (500 μg/mL) selection for 10 days. Transfected MM cells were selected, and expression of C/EBPβ and IRF4 was detected by Western blotting with anti-C/EBPβ and anti-IRF4 antibody. β-Actin was used as a loading control. (D top panel) Expression of IRF4 in MM cell lines and primary MM cells was detected by Western blotting as described in “Western blot analysis.” (D bottom panel) Cells were cultured in 1% FBS with or without IL-6 for 48 hours, and analyzed for induction of IRF4 protein expression. β-Actin was used as a loading control for all samples. Vertical lines have been inserted to indicate a repositioned gel lane.