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. 2013 Feb 28;8(2):e57795. doi: 10.1371/journal.pone.0057795

Figure 4. RSUME267 and RSUME195 similar actions on the SUMO pathway.

Figure 4

A. 1 µg of recombinant RSUME195 or RSUME267 was co-precipitated by GST-Ubc9 in vitro. Pull down experiments were performed and the samples were subjected to Western blot. To compare, the ratio between input and pull-down intensities measured by Image-J Software was calculated. As internal control, to detect unspecific binding, GST alone was used to pull-down any RSUME isoform. Ø, control elution extract purified from E.coli transformed with the empty pQE30 vector. B. 300 ng HA-SUMO-1, expression vector was co-transfected with 500 ng of V5-RSUME195 or V5-RSUME267 and V5-Ubc9 expression vectors. 48 h post-transfection, cell extracts were subjected to western blot with anti-HA to detect sumoylated proteins. RSUME expression was confirmed with anti-RSUME antibodies. C. Sumoylation assay of Topoisomerase I (TOPOI) to test enhancer properties of RSUME isoforms. Experiments were performed in triplicates.