Fig. 1

HIV-1 Gag interacts with human ribosomal protein RPL7. a Photograph of a plate with diploid yeast cells expressing Gal4BD alone or fused to either NCp7, Gag or GagΔNC (pGBKT7 construct) and Gal4AD alone or in fusion to RPL7 (pActII construct). Yeasts were plated on minimal media lacking histidine, leucine and tryptophan and incubated during 6 days at 30 °C. Growth in the absence of histidine reveals the interaction between the hybrid proteins. Note that NCp7 and Gag interact with RPL7 while marginal interaction was observed between Gag∆NC and RPL7. b Gag co-precipitates with endogenous RPL7. Lysates from HeLa cells transfected with pcDNA (lanes 1 and 3) or a plasmid expressing Gag (lanes 2 and 4) were subjected to IP (1 mg of total protein) using anti-p24 antibody. 20 µg of total protein (input) and resuspended beads were analyzed western blot with anti-p24, anti-RPL7 and anti-GAPDH antibodies revealed by protein A-HRP. RPL7 signal was observed in lane 4 showing that RPL7 was captured by Gag. In contrast, no RPL7 signal was observed in the pcDNA tranfected cells (lane 3) or in the assay where the beads were incubated in absence of anti-p24 antibody (lane 5). c Flag–RPL7 co-precipitates Gag-eGFP. HeLa cells were transfected with pcDNA (lanes 1 and 5), Gag-eGFP (lanes 2 and 6), Flag–RPL7 (lanes 3 and 7) or co-transfected with Flag–RPL7 and Gag-eGFP (lanes 4, 8 and 9). IP was performed with 1.5 mg of total protein with a mouse anti-Flag antibody. 20 µg of input (lanes 1–4) and IPs (lanes 5–8) were resolved by 10 % SDS-PAGE and analyzed by immunostaining using an antibody directed against Flag to detect RPL7, eGFP to detect Gag, and GAPDH as a loading control. RPL7 is able to co-immunoprecipitate Gag-eGFP in HeLa cells (lane 8). No unspecific binding between Flag–RPL7 and beads was observed in the control without anti-flag antibody (lane 9)