Figure 2.
Modification of SARS‐CoV N protein by SUMO‐1. (A) Analysis of sumoylation of N protein by Western blotting. Cell lysates prepared from HeLa cells overexpressing either the Flag‐tagged N protein alone (lanes 1 and 2) or together with SUMO‐1 (lanes 3, 4, 5, and 6) were prepared either in the presence (lanes 2, 3 and 5) or absence (lanes 1 and 4) of the isopeptidase inhibitors IAA and NEM or by direct lysis in preheated SDS loading buffer (lane 6). The polypeptides were separated by SDS–PAGE under either reducing (lanes 1–3) or non‐reducing (lanes 4–6) conditions and analyzed by Western blotting with anti‐Flag antibody. The three major isoforms of N protein are indicated by brackets and the major SUMO‐1 modified form of N protein is indicated by asterisks. Numbers on the left indicate molecular masses in kilodaltons. (B) Analysis of sumoylation of N protein by immunoprecipitation and Western blotting. Total cell lysates were prepared, in the presence of the isopeptidase inhibitors IAA and NEM, from HeLa cells expressing SUMO‐1 (lanes 1 and 4), Flag‐N (lanes 2 and 5), and Flag‐N + SUMO‐1 (lanes 3 and 6), and immunoprecipitated with either anti‐Flag (lanes 1, 2 and 3) or anti‐SUMO‐1 antibody (lanes 4, 5, and 6). The immunoprecipitated proteins were separated by SDS–PAGE under nonreducing conditions and analyzed by Western blotting with anti‐Flag antibody. The major SUMO‐1 modified forms of N protein are indicated by asterisks, and the immunoglobulin is indicated by Ig.