Fig. 3. HIV-1 IN and JPO2 compete for binding to the p75 IBD. (A) The p75 IBD is necessary and sufficient for the interaction with JPO2. Lanes 1 and 2; input levels of BSA and ³⁵S-labeled JPO2, respectively. Lanes 3-9: GST, GST-p75 or the indicated p75 deletion mutant pre-bound to GS beads was incubated with ³⁵S-labeled JPO2. BSA (10 µg) was included in each reaction as an internal specificity control. Bound proteins separated by SDS-PAGE were stained with Coomassie Blue (top panel); JPO2 was detected by autoradiography (bottom panel). (B) HIV-1 IN competes with JPO2 for binding to p75 IBD. Lanes 1-4: input BSA, IN-His₆, p75 and ³⁵S-labeled JPO2, respectively. Lanes 5-10: His₆-tagged HIV-1 IN was incubated with p75 and/or JPO2 in the presence of Ni-NTA beads and BSA, and bound proteins fractionated by SDS-PAGE were detected by staining with Coomassie Blue (top panel) or autoradiography (bottom panel). Migration positions of molecular mass markers, IN-His₆, p75, BSA and JPO2 are indicated.