Fig. 3

The NC domain of Gag mediates Gag–RPL7 interaction in an RNA independent manner. a1 Gag and truncated Gag constructs used in the present study. Numbers on the right correspond to numbers of lanes of a2. a2 The NC domain of Gag is important for the RPL7–Gag interaction. HeLa cells were not transfected (NT) or transfected with pcDNA or plasmids encoding either Gag (construct 1), Gag∆NC (construct 2), Gag∆ZF1 (construct 3), Gag∆ZF2 (construct 4), GagRAPAAA (construct 5) and Gag∆ZF1ZF2 (construct 6). IP was performed with 1 mg of total protein and with an anti-p24 antibody. 20 µg of cell lysate (input) or IP resuspended samples were resolved by 10 % SDS-PAGE and analyzed by immunoblotting using antibodies against p24, RPL7 and GAPDH revealed by protein A-HRP, anti-rabbit HRP conjugate or anti-mouse HRP conjugate, respectively. Nonspecific binding was not observed in the control without antibody (lane 7). b NCp7-RPL7 interaction is RNA independent. Cell lysate from HeLa cells transfected with empty pcDNA (lanes 1, 4) or plasmids coding for eGFP (lanes 2, 5) or NCp7-eGFP (lanes 3, 6). Cell lysates were immunoprecipitated with an anti-eGFP and the immunoprecipitated material was examined by western blot using anti-eGFP, RPL7 and GAPDH antibodies. Lane 7 cell lysates expressing NCp7-eGFP were treated with RNase before immunoprecipitation. Lane 8 cell lysate expressing NCp7-eGFP was incubated with protein A beads but without anti-eGFP antibody. c Gag–RPL7 interaction is RNA independent. Cell lysate from HeLa cells transfected with Gag was treated with RNase. After RNase treatment, cell lysate was immunoprecipitated with anti-p24 antibody, and the immunoprecipitate was analysed by western blot using anti-p24 and anti-RPL7 antibodies