Figure 1: SENP2 is able to cleave SUMO-2/3 from endogenous proteins in harsh buffer conditions.

(a) HeLa cells were lysed in 8 M urea, homogenized and subsequently diluted to reduce the concentration of urea as indicated. Lysates were treated with SENP1, SENP2 or mock treated. After protease treatment, lysates were size-separated by SDS–PAGE, transferred to membranes, probed using a SUMO-2/3 antibody and visualized using chemiluminescence. The experiment was performed in biological triplicate. (b) As in a, and subsequently films were scanned and the amount of conjugated SUMO-2/3 was quantified and plotted against urea concentration. n=3, error bars represent s.e.m., asterisks indicate a significant difference as compared with control with P<0.01 by two-tailed Student’s t-test. (c) As in a, but probed using a SUMO-1 antibody. SUMOylated RanGAP1 is indicated with an arrow. (d) As in b, but using a SUMO-1 antibody. (e) As in a, but probed using a ubiquitin antibody. (f) As in b, but using a ubiquitin antibody. (g) As in a, but with total protein content visualized using Ponceau-S staining.