FIGURE 1.

GANP interacts with AID in vivo and in vitro. A, AID co-IP with GANP. Lysates from COS-7 cells, transfected with GFP-GANP, AID, GFP-GANP + AID, or GFP-alone constructs, were immunoprecipitated using anti-GFP Ab. Immunoblotting (IB) was carried out with anti-GFP Ab, anti-AID Ab, or anti-β-actin Ab by IP-Western blot analysis. Specific association in the IP with the anti-GFP Ab was confirmed by comparing with experiments using the control IgG (bottom panel). Western blots for whole cell lysates (WCL) are shown. B, reverse IP-Western blot analysis was performed by IP with anti-AID Abs followed by immunoblot with anti-GFP Ab or anti-AID Ab. The specific signal representing 240-kDa GFP-GANP was detected in immunoprecipitates using both mouse anti-AID Abs (M mAb) and rabbit anti-AID Abs (R mAb). The anti-AID (M) mAb contains the IgL band (25-kDa) used for IP as indicated with an asterisk. C, the WCL from COS-7 cells transfected with GFP-GANP and AID constructs was incubated for 10 min with either RNase A (5 μg/ml (+R) or 250 μg/ml (++R)) or DNase I (10 units (+D) or 50 units (++D)) at 4 °C or 37 °C and then immunoprecipitated with anti-GFP Ab followed by immunoblot with anti-GFP or anti-AID (M) Ab. D, interaction of endogenous GANP and AID was examined in Ramos B-cells by co-IP. The asterisk shows the IgL band. E and F, FLAG-GANP and AID proteins were synthesized in vitro using a WGE cell-free system, and coimmunoprecipitated using anti-AID (M) Ab (E) or anti-FLAG Ab (F). Controls for specificity of co-IP experiments using mouse (M) IgG are shown in the bottom panels (E and F). G, the interaction between WGE-produced GANP and AID was still preserved after treatment with RNase A or DNase I. The data were determined from three independent experiments.