Effect of HIV-1 integrase on the chromatin binding activity of LEDGF/p75. (a) The chromatin binding strength of LEDGF/p75 WT and ΔPWWP was determined by the salt extraction method using LEDGF/p75-deficient HEK293T cells stably expressing these LEDGF/p75 proteins alone or together with Myc-tagged HIV-1 integrase. Immunoblots show the amount of LEDGF/p75 extracted from chromatin at different concentrations of NaCl as detected with an anti-LEDGF Mab. T represents a total cellular lysate obtained by boiling the cells in Laemmli buffer. (b) The intensity of different immunoblot bands in panel (a) was quantified by densitometry analysis and expressed as percentage of the intensity of the bands corresponding to the total cellular lysate (T). Errors bars were calculated using two different experiments. (c) Chromatin binding assay. The subcellular distribution of LEDGF/p75 WT or ΔPWWP mutant was evaluated by immunoblotting with an anti-LEDGF Mab in cells stably expressing these LEDGF/p75 proteins alone or together with Myc-tagged HIV-1 integrase. S1 and P2 are non-chromatin bound fractions; P1 and S2 are chromatin-bound fractions, and T is a total cellular lysate. The S1 fraction was obtained by lysing the cells in CSK I buffer containing 150 mM NaCl.