Correlation between RAG proteins expression and new light chain expression. (A) Effect of PMA that has been shown to moderate RAG expression was examined. HTD8 cells were treated with (+) or without (−) 50 ng/mL PMA. Cells were lysed in SDS lysis buffer; 50 μg/mL protein from each sample was fractionated by 10% SDS-PAGE, and RAG-1 and RAG-2 proteins were detected by immunoblotting. (B) Lambda light chain expression pattern in HTD8 cells treated with PMA. The pattern of λ light chain secretion from HTD8 cells cultured with (+) or without (−) PMA in a 96-well culture plate for 4 weeks and eight wells were randomly chosen, and the culture supernatants were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and λ light chains were detected by immunoblotting. (C) The 32-kD original λ chain producing subclones generated from the PMA treatment was cloned via the limiting dilution method, further cultured for 2 weeks in a 96-well plate, and eight randomly chosen wells were assayed for λ light chain production by immunoblotting. (D) The expression of RAG-1 and RAG-2 proteins in the cells indicated above each lane was assessed by immunoblotting.