Characterization of IN mutants binding to Ini1 and LEDGF/p75.A. Interaction of the INwt and mutant with Ini1. Different IN-YFP plasmids, as indicated, were co-transfected with pCGN-HA-Ini1 in 293T cells. At 48 hrs post-transfection, cells were lysed and immunoprecipitated with rabbit anti-GFP antibody and subjected to WB with anti-HA antibody to measure the amount of co-precipitated Ini1 (upper panel). The same membrane was stripped and blotted with anti-GFP antibody to detect IN-YFP and YFP expression (middle panel). The unbound Ini1 was also checked by sequential anti-HA IP followed by WB with the same antibody (lower panel). B. Lethal phenotype-defective IN mutants do not bind to LEDGF/p75. Different IN-YFP expressors were co-transfected with T7-LEDGF expressor in 293T cells. Cells were lysed 48 hrs post-transfection, whole-cell protein extracts were immunoprecipitated with rabbit anti-GFP followed by a WB using mouse anti-T7 HRP-conjugated antibody to detect the co-precipitated T7-LEDGF (upper panel). Also, the expressions of IN-YFPs were detected using an anti-GFP HRP-conjugated antibody (middle panel). The unbound T7-LEDGF in the cell lysate was also checked by sequential IP with anti-T7 antibody followed by a WB with the same antibody (lower panel).