SDS-PAGE and Western Blotting

For Western blotting, cells were lysed in Laemmli buffer (2% SDS, 5% 2-β-mercaptoethanol, 5% glycerol, 62.5 mM Tris-HCl pH 6.4, 0.002% bromophenol blue), briefly sonicated to shear DNA and heated to 37 °C for 10 min. Proteins in cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane for Western blotting. Membranes were blocked in 5% w/w non-fat milk powder in PBS-T. Primary antibodies used for blotting were: SUMO1 (rabbit monoclonal, Abcam ab133352, 1:1000), SUMO2/3 (rabbit monoclonal, Abcam ab109005, 1:1000), Ubc9 (rabbit monoclonal, Abcam ab75854, 1:3000), GFP (mouse monoclonal, Abcam ab1218, 1:500), alpha-tubulin (mouse monoclonal, Cell Signaling Technologies clone DM1A, 1:5000), RanGAP1 (rabbit monoclonal ab92360, 1:1000), beta-tubulin (mouse monoclonal, Invitrogen PAS-16863, 1:3000), myc (mouse monoclonal, Cell Signaling Technologies clone 9B11, 1:1000). For SUMO1 pulldowns, cells were lysed in 1% triton X-100, 0.1% sodium-dodecyl sulphate, 150 mM NaCl, 25 mM HEPES (pH 7.4). 20 mM NEM was either included or omitted from the lysis buffer dependent on experimental conditions. SUMO1 pulldowns were performed as previously described²⁴. For protein detection, membranes were incubated with either horseradish peroxidase (HRP)-conjugated (1:10000) or IRDye-conjugated (1:20000) secondary antibodies (both from LI-COR). HRP signal was visualised by enhanced chemiluminescence, and IRDye signal by fluorescence, both using an Odyssey Fc Imager (LI-COR).