Figure 5

LEDGF/p75 is not required for chromatin binding of IN. A). Transient knockdown of LEDGF/p75 by siRNA had no effect on IN nuclear localization. 293T cells were transfected with either 20 nM negative control (NC) siRNA or 20 nM si-LEDGF PSIP1HSS146003 for 24 h before transfection with CMV-YFP-IN wild type. At 48 h post-transfection, cells were fixed, permeabilized and detected for YFP-IN and LEDGF/p75 expression by using anti-GFP or anti-LEDGF antibodies. The nuclei were stained with DAPI. B). Analysis of chromatin binding affinity of IN on LEDGF/p75 knockdown cells. The lentiviral shRNA-mediated LEDGF/p75 stable knockdown 293T cells were transfected with 20 nM si-LEDGF for 48 h and further transfected with YFP-IN wild type or mutant V165A and were analyzed for its chromatin binding affinity. In parallel, cells were either mock-transfected or transfected with negative control siRNA to study chromatin binding of YFP-IN wild type. The chromatin bound and non-chromatin-bound fractions of YFP-IN wild type or V165A were showed as indicated. The LEDGF/p75 expression level in each sample was verified by WB with anti-LEDGF antibody. Endogenous beta-actin was used for normalization of sample loading.