Fig. 4

IN SIMs differentially regulate their interaction with LEDGF/p75 and Ku70. a Schematics of various plasmid constructs encoding GFP-INwt, T7-LEDGF/p75, T7-Ku70 and ProLabel-Ku70 (PL-Ku70). b The interaction of T7-LEDGF/p75 with GFP-INwt or mutants (3KR, 3VI, M1, M2, M3, M1 + M2, M1 + M3 and M2 + M3) was analyzed by co-IP assay. The GFP-IN-bound T7-LEDGF/p75 was detected by immunoprecipitation of the cell lysates with an anti-GFP antibody and immunoblotting with an anti-T7-HRP antibody (upper panel). The expression of GFP, GFP-INwt/mut and T7-LEDGF/p75 are shown in the middle and lower panels. The results are representative of three independent experiments. c The interaction of Ku70 with INwt/mutant was detected by co-IP assay. Left panel: The upper panel showed the bound T7-Ku70 in each sample. Two percent of cell lysates were used to detect the expression of GFP, GFP-INwt/mut and T7-Ku70 by WB (middle panel and lower panel). Middle and right Panels: The interaction of ProLabel-Ku70 (PL-Ku70) with GFP-INwt/mut was detected by chemiluminescent co-IP assay. The chemiluminescent signals from IN-bound PL-Ku70 present in the complexes were measured using the ProLabel Detection Kit II and valued as relative luminescence units (RLU). The results are representative of two independent experiments. Expression levels of PL-Ku70 and GFP-IN wt/mut or GFP alone in each sample were analyzed by anti-Ku70 and anti-GFP-HRP antibodies (right panel)