Supplementary Figure 5: The glutamate in the SUMOylation consensus motif of RanGAP1 is required for efficient SUMOylation, and Tel contains an inverted ExK SUMOylation site at Lys302.
From: Uncovering global SUMOylation signaling networks in a site-specific manner
(a) Immunoblot analysis (IB) of total lysates from HeLa cells or HeLa cells expressing His10-SUMO-2 which were transfected with wild-type (WT) HA-RanGAP1, HA-RanGAP1 E526D, or a SUMOylation-deficient RanGAP1 (ΔGL). On the left side, “S2” indicates SUMO-modified RanGAP1, and asterisks indicate non-specific bands. Ponceau-S staining is shown as a loading control. The experiment shown was replicated in biological duplicate.
(b) Similar to (a), but using U2-OS cells. The experiment shown was replicated in biological duplicate.
(c) Immunoblot analysis of His10-pulldown samples (PD) from HeLa cells or HeLa cells expressing His10-SUMO-2 which were transfected with the indicated HA-Tel constructs. On the left side, “S2” indicates SUMO-modified Tel, and asterisks indicate non-specific bands. SART1 is shown as a pulldown control, and Ponceau-S staining is shown as a pulldown and loading control. HA-Tel-K11R was used to monitor SUMOylation of K300, because Tel harbors a dominant major SUMOylation site at lysine-117. The experiment shown was replicated in biological triplicate.
(d) Total lysates corresponding to (c), analyzed by SDS-PAGE and immunoblotting. Ponceau-S staining is shown as a loading control. The experiment shown was replicated in biological triplicate.
