Figure 4
From: Targeted editing of the PSIP1 gene encoding LEDGF/p75 protects cells against HIV infection
LEDGF D366N mutant is uncoupled from HIV-IN but interacts with other binding partners. (a–c) In vivo co-localization of the LEDGF D366N and cellular binding partners. Wild-type HEK293T, LEDGF KO and LEDGF D366N cells were transfected with flag-tagged IWS1, JPO2 and HIV-IN (panels a, b and c, respectively) and fixed 30 hrs later. Fluorescence microscopy to assess localization of proteins: LEDGF/p75 and LEDGF D366N were detected using αLEDGF480–530 (shown in green), whereas the transfected flag-tagged proteins were detected with αFlag Ab (shown in red). (a,b) LEDGF WT (top panel) and LEDGF D366N (lower panel) co-localizes with IWS1 and JPO2. (c) LEDGF D366N mutant did not co-localize with HIV-IN (lower panel), while in HEK293T WT cells HIV-IN is retained in the nucleus by binding to LEDGF/p75 (top panel). Scale Bar: 10 µm. (d) Co-immunoprecipitation of LEDGF/p75 and LEDGF D366N by cellular binding partners. HEK293T_LEDGF KO cells were transfected with either WT or D366N mutant LEDGF plasmid along with either one of the plasmids encoding Flag-tagged binding partners: IWS1, JPO2 or HIV IN. Cells were lysed 24 h later and lysates were incubated with anti-FLAG® M2-agarose affinity resin to capture the binding partner protein, which was then visualized by Western blot using αFlag Ab and αLEDGF480–530.
