Figure 1. Interaction of LEDGF/p75 with the FACT complex.
(a) Chromatin-bound proteins were isolated from TL3 and TL3 LEDGF/p75 WT cells by DNase treatment and FLAG-tagged LEDGF/p75 was immunoprecipitated from this subcellular fraction with an anti-FLAG mAb antibody. The presence of the FACT complex (hSpt16 and SSRP1) was evaluated in the immunoprecipitated proteins by immunoblotting with specific antibodies. (b) Quantitative confocal microscopy co-localization of LEDGF/p75 with SSRP1. Panels 1 and 2 are controls and represent the co-localization of HIV integrase with LEDGF/p75 mutant lacking the integrase-binding domain (ΔIBD) or LEDGF/p75 wild type (WT). LEDGF/p75-deficient HEK293T cells stably expressing eGFP-tagged HIV-1 integrase were transiently transfected with LEDGF/p75ΔIBD or WT. LEDGF/p75 proteins were detected with an anti-LEDGF antibody. The lower panels represent the co-localization of endogenous LEDGF/p75 with endogenous SSRP1 or Pol II in HeLa cells after immunostaining with specific antibodies. (c) Quantification of co-localization data in (b). Standard deviations indicated co-localization values found in ten different cells randomly selected from a field representative of ten different random areas of the microscope slide. (d) HEK293T cells were co-transfected with plasmids expressing: FLAG-tagged LEDGF/p75 and Myc-tagged SSRP1 WT (lane 1), FLAG-tagged LEDGF/p75 and Myc-tagged SSRP1ΔNTD (lane 2), or Myc-tagged SSRP1 WT and an empty plasmid (lane 3). Samples were analyzed in the same gel, the line dividing lanes 2 and 3 indicate that lanes in between were removed for comparison in this figure. In 1d, (*) indicates degradation products of SSRP1