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Figure 6 | BMC Biochemistry

Figure 6

From: Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome

Figure 6

CSN1 inhibits SAP130-Cul2 interaction and SAP130 associated E3 activity. (A) In vitro translated HA-SAP130 in RRL was incubated with GST-Nedd8 (1 μg) and GST-CSN1 (0.5 μg or 1.0 μg) or GST (1.0 μg). The RRL input and HA-IP were analyzed by anti-Cul2 blot. (B) Whole cell extract from Flag-SAP130 stable cells were either left untreated or pretreated with GST (2 μg), GST-CSN1 (1 μg and 2 μg) or purified CSN complex from porcine spleen (CSN com). Flag immunocomplex was subsequently isolated and used for in vitro E3 activity assay. Polyubiquitin chain was detected by anti-ubiquitin blot. Equal amount of Flag-SAP130 immunocomplex was confirmed by anti-Flag blot. Cul2 blot of the pretreated extract showed reduced neddylation after incubation with CSN complex but not GST-CSN1.

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