Identification of LEDGF/p75-binding sites within IN CCD. A). The CMV-YFP-INwt/mut or CMV-YFP plasmid was co-transfected with SVCMVin-T7-LEDGF expressor in 293T cells. After 48 h of transfection, 90% cells were lysed and subjected to co-IP assay. The IN bound T7-LEDGF/p75 was precipitated by using rabbit anti-GFP antibody and detected by WB using mouse anti-T7 antibody (upper panel). 10% cells were lysed with 0.5% NP-40, directly loaded on 10% SDS-PAGE gel and probed with anti-T7 or anti-GFP antibody to detect T7-LEDGF or YFP-IN expression (middle or lower panel). B). The intensity of protein bands was densitometrically determined. Results were expressed as the ratio of bound T7-LEDGF/p75 expression (mutants/wild-type) which was normalized by total input. Binding affinity to LEDGF/p75 of YFP-IN wild type was arbitrarily set as 100%. Results are representative of two independent experiments.