Gag-Env interaction analysis using different combinations of prokaryotic and eukaryotic expressed proteins. (A) Schematic outline of the prokaryotic PFV Gag expression constructs. On top the outline of the wild type full-length PFV Gag coding sequences are shown as grey boxes with different structural and functional domains indicated as in the legend of Figure 1. (B-C) Representative Western blot analysis of pull-down assays using various combination of lysates of 293T cells (euk.) containing different GST-tagged Env or EGFP-HH-tagged Gag proteins or affinity purified proteins expressed in E. coli (prok.) as indicated. Samples of Gag expressing cell lysates corresponding to 10% of the input (input) for the pull-down assay are shown to the right, samples of proteins eluted by boiling in SDS-PAGE loading buffer from pelleted glutathione-sepharose beads (pull-down) are shown to the left. Prey (Gag and ctrls) proteins were detected using polyclonal antibodies specific for PFV Gag (α-Gag), bait (Env) proteins using a polyclonal anti-GST serum (α-GST). Preys: 1–310: pcziPG4 1–310 CLEGHH; 1–310 MH: pET11a Gag 1–310 MH; 1–310 MH L17A: pET11a Gag 1–310 MH L17A; 1–310 MH L17S: pET11a Gag 1–310 MH L17S; 41–310 MH: pET11a Gag 41–310 MH; MH: pET11a MH. Baits: e-wt: pCAG Env1-60 CLGST; e-W/A: pCAG Env1-60 CLGST W/A; p-wt: pET11a Env1-60 CLGST; p-W/A: pET11a Env1-60 CLGST W/A.