Skip to main content
. Author manuscript; available in PMC: 2013 Sep 1.
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. 3.

Fig. 3

Sumo1 conjugation motif resided in the carboxyl terminus of LEDGF and Sumoylated. (A) Schematic representation of full-length LEDGF protein showing the putative Sumo1 conjugation motif(s) as predicted by SUMOplot (Abgent). The position of modifiable lysine (K) site(s) in each Sumo1 site is indicated by number. GST-linked full-length LEDGF -or NH2 - or COOH- LEDGF used in assay are shown as Full, N and C, respectively. The amino acid sequence of LEDGF was analyzed to identify possible Sumoylation sites. Full and deleted NH2-and COOH-LEDGF constructs (N, 1-250aa or C, 170-530aa) were generated using PCR with LEDGF sense and antisense primers and cloned into pGEX-2T vector. Recombinant protein was purified and used for assay. (B) C-terminal LEDGF was targeted by Sumo1 conjugation, while NH2 terminus was not. To analyze which region of LEDGF was Sumoylated, N-terminal (N, 1-250aa) and C-terminal (C, 170-530aa) deletion constructs of LEDGF were subjected to in vitro Sumoylation assay. Western blot analysis was conducted with LEDGF antibody. Sumoylated C-terminal LEDGF band with retarded mobility (B, lane 3); NH2–terminal, lanes, 1 and 2. (C) Western analysis image showing the expression of purified recombinant NH2-and COOH-LEDGF protein immunostained with anti-GST antibody.