Figure 9: Confirmation of PRISM-identified SUMO target proteins.
From: System-wide identification of wild-type SUMO-2 conjugation sites
(a) HeLa cells, HeLa cells stably expressing His10-SUMO-2, U2OS cells and U2OS cells stably expressing His10-SUMO-2 were harvested and lysed. SUMOylated proteins were purified using nickel-affinity chromatography. Total lysates and SUMO-enriched fractions (PD: His) were size-separated by SDS–PAGE, transferred to membranes, probed using the indicated antibodies and visualized using chemiluminescence. Asterisks indicate non-specific bands, and arrows indicate SUMOylated proteins. The experiment was performed in biological duplicate. (b) Controls for a, with Ponceau-S staining of total lysates as a loading control, and SUMO-2/3 immunoblot analysis of enriched fractions as a pulldown control. (c) As in a, with all cell lines incubated at 43 °C for 1 h or mock treated prior to lysis. SILAC heat shock response ratios (log2) are indicated. Asterisks indicate non-specific bands, and arrows indicate SUMOylated proteins. The experiment was performed in biological duplicate. (d) Controls for c, with Ponceau-S staining of total lysates as a loading control, and SUMO-2/3 immunoblot analysis of enriched fractions as a pulldown control.
