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Published in final edited form as: FEBS J. 2012 Jul 16;279(17):3048–3070. doi: 10.1111/j.1742-4658.2012.08686.x

Fig. 5 — LEDGF was modified by Sumo1 in vivo, and conserved lysine (K) 364 in the IBD domain of COOH-terminal is a Sumo1 conjugation motif. Cells were co-expressed with pEGFP-Sumo1 and pEGFP-LEDGF or pEGFP-K364R along with pCMV-Ubc9. Cellular extracts were processed for immunoprecipitation (IP) using anti-LEDGF monoclonal antibody. (A) The membrane was immunoblotted with anti-Sumo1 antibody: endogenous Sumoylated LEDGF (~87kDa), endogenous LEDGF Sumoylated with exogenous EGFP-Sumo1 (~115kDa; EGFP-Sumo1+LEDGF) and exogenous LEDGF Sumoylated with exogenous EGFP-Sumo1 (~143kDa; EGFP-LEDGF+EGFP-Sumo1). (B) The same membrane was immunoblotted with anti-LEDGF antibody: unSumoylated endogenous LEDGF (~75kDa), unSumoylated EGFP-LEDGF or K364R (~105 kDa), endogenous Sumoylated LEDGF (~87kDa), and endogenous LEDGF Sumoylated with exogenous EGFP-Sumo1 (~115kDa; *EGFP-Sumo1+LEDGF) and exogenous EGFP-LEDGF plus exogenous EGFP-Sumo1 (~143kDa; **EGFP-LEDGF+EGFP-Sumo1). (C) The same membrane was immunoblotted with anti-GFP antibody: EGFP-LEDGF or K364R (~105 kDa), Endogenous LEDGF Sumoylated with exogenous EGFP-Sumo1 (~115kDa; *EGFP-Sumo1+LEDGF) and exogenous EGFP-LEDGF Sumoylated with EGFP-Sumo1 (~143kDa; **EGFP-LEDGF+EGFP-Sumo1). Sumoylated form of Mutant LEDGF (K364R) could not be detected; indicating Lysine (K) at 364 is the major Sumoylation site in LEDGF protein.

(D) Immunofluorescence images showing localization of LEDGF and its mutant form, EGFP-K364R. Cells were transfected with wild type pEGFP-LEDGF (left panel) or mutant LEDGF pEGFP-K364R (right panel) and fluorescence images of live cells were recorded after 24 h transfection under inverted fluorescence microscope (Nikon Eclipse Ti-U). E, Western analysis of cellular extract obtained from LECs transfected with pEGFP-LEDGF or pEGFP-K364R following equalization with GFP (O.D. at Ex485/Em530).

Fig. 5 — LEDGF was modified by Sumo1 in vivo, and conserved lysine (K) 364 in the IBD domain of COOH-terminal is a Sumo1 conjugation motif. Cells were co-expressed with pEGFP-Sumo1 and pEGFP-LEDGF or pEGFP-K364R along with pCMV-Ubc9. Cellular extracts were processed for immunoprecipitation (IP) using anti-LEDGF monoclonal antibody. (A) The membrane was immunoblotted with anti-Sumo1 antibody: endogenous Sumoylated LEDGF (~87kDa), endogenous LEDGF Sumoylated with exogenous EGFP-Sumo1 (~115kDa; EGFP-Sumo1+LEDGF) and exogenous LEDGF Sumoylated with exogenous EGFP-Sumo1 (~143kDa; EGFP-LEDGF+EGFP-Sumo1). (B) The same membrane was immunoblotted with anti-LEDGF antibody: unSumoylated endogenous LEDGF (~75kDa), unSumoylated EGFP-LEDGF or K364R (~105 kDa), endogenous Sumoylated LEDGF (~87kDa), and endogenous LEDGF Sumoylated with exogenous EGFP-Sumo1 (~115kDa; *EGFP-Sumo1+LEDGF) and exogenous EGFP-LEDGF plus exogenous EGFP-Sumo1 (~143kDa; **EGFP-LEDGF+EGFP-Sumo1). (C) The same membrane was immunoblotted with anti-GFP antibody: EGFP-LEDGF or K364R (~105 kDa), Endogenous LEDGF Sumoylated with exogenous EGFP-Sumo1 (~115kDa; *EGFP-Sumo1+LEDGF) and exogenous EGFP-LEDGF Sumoylated with EGFP-Sumo1 (~143kDa; **EGFP-LEDGF+EGFP-Sumo1). Sumoylated form of Mutant LEDGF (K364R) could not be detected; indicating Lysine (K) at 364 is the major Sumoylation site in LEDGF protein.

(D) Immunofluorescence images showing localization of LEDGF and its mutant form, EGFP-K364R. Cells were transfected with wild type pEGFP-LEDGF (left panel) or mutant LEDGF pEGFP-K364R (right panel) and fluorescence images of live cells were recorded after 24 h transfection under inverted fluorescence microscope (Nikon Eclipse Ti-U). E, Western analysis of cellular extract obtained from LECs transfected with pEGFP-LEDGF or pEGFP-K364R following equalization with GFP (O.D. at Ex485/Em530).