Identification of the AID protein by specific antibodies. (A) Northern blot analysis of endogenous AID expression in 2 BL (ODHI I, ODHI III) and 2 DLBCL (Ly10, SUDHL6) cell lines. An approximately 2.7-kb message, corresponding to the AID transcript, can be detected in ODHI I and Ly10 but not in ODHI III and SUDHL6. Filters were stripped and sequentially hybridized with probes for BCL6, as marker of differentiation stage, and GAPDH as control for loading. (B) Western blot analysis of the same cell lines with anti-AID rabbit polyclonal Abs shows a specific band of the predicted 24-kDa molecular weight in ODHI I and Ly10, but not in ODHI III and SUDHL6, consistent with the RNA data in panel A. Immunoblotting with anti-BCL6 (N3) and anti-β-actin Abs is shown in the bottom panels. (C) Western blot analysis of AID expression in H1299 and Ramos cells, stably transduced with pBABE retroviral vectors (H1299-V, Ramos-V) or with vectors expressing an AID-Flag-HA protein (H1299-AID-FH and Ramos-AID-FH). Both the rabbit polyclonal antiserum (poly) and the mouse monoclonal (7E7) Abs were used (top 2 panels). An approximately 27-kDa band corresponding to the size of the exogenous protein (exo) is detected in lysates from AID-FH-transduced cells (lanes 2 and 4) but not in cells transduced with the control vector (lanes 1 and 3). In the B-cell line Ramos, the endogenous AID protein (endo) can also be seen (lanes 3 and 4). Membranes were stripped and sequentially probed with anti-HA, which recognizes the exogenous protein, and with anti-β-actin as control for loading (bottom 2 panels).