(A) Experimental design. CH12 cells were transduced with empty vector (pEF) or vectors to express forward/sense or reverse/anti-sense switch α RNA (pEF-SαF or pEF-SαR, respectively). Transduced cells were sorted, infected with scrambled control shRNA or shRNA to knockdown DBR1, and stimulated with CIT.
(B–C) Expression of exogenous SαF rescues AID localization to Sα at the endogenous IgH locus, but not to the non-complementary Sμ. ChIP was performed on cells 48 h post stimulation by immunoprecipitation with anti-AID antibodies. Sα (B) and Sμ (C) DNA in ChIP samples was quantified by qPCR, and normalized to input and scrambled control.
(D) Expression of exogenous SαF does not rescue CSR in DBR1 knockdown cells. CSR to IgA was assayed 72 h after stimulation by flow cytometry. Data in (B)–(D) represent the mean of three independent experiments ± SD. *p<0.05; NS, p=not significant, p≥0.05.
See also Figure S6.