Fig. 2. LIN-1 is covalently-modified by SUMO-1. (A) Extracts from yeast expressing LA:LIN-1(1-252) alone (-) or with His- and FLAG-tagged SUMO1/Smt3 (HF-SUMO) (+) were subjected to metal affinity chromatography. Bound proteins were separated by SDS-PAGE and immunoblotted (IB). An anti-FLAG antibody detected all proteins modified by HF-SUMO; an anti-LA antibody detected LA:LIN-1(1-252) complexes. The arrows indicate high molecular weight forms of LA:LIN-1(1-252) that appear to be covalently modified by one or multiple HF-SUMO moieties (14 kDa), and may also contain endogenous SUMO1/Smt3 (11 kDa). Bands present in lanes 3 and 4 are a cross-reactive endogenous yeast protein that was present in strains lacking LA:LIN-1 (data not shown) and 60 kDa unmodified LA:LIN-1(1-252) that bound the affinity matrix in a Ni²⁺-independent manner (data not shown). Molecular weight markers (in kDa) are indicated. (B) Extracts from Sf9 cells that were not infected (Mock) or infected with viruses that express GST:LIN-1 alone (-) or with His- and FLAG-tagged C. elegans SMO-1 (HF-SUMO) (+) were subjected to glutathione sepharose affinity chromatography to purify the GST fusion proteins. Bound proteins were separated by SDS-PAGE and immunoblotted. An anti-GST antibody detected all LIN-1 species; an anti-FLAG antibody detected LIN-1 that was covalently modified by HF-SUMO. Arrows indicate sumoylated isoforms of LIN-1 (lanes 3 and 7) that were absent in extracts containing mutant GST:LIN-1(1-64; 9-16A) (lanes 4 and 8). (C) Extracts from Sf9 cells were analyzed as in B. Arrows indicate sumoylated isoforms of LIN-1 (lane 5) that were absent in extracts containing mutant GST:LIN-1(1-64; K10A) (lane 6).