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. 2007 Jan 29;104(6):1805–1810. doi: 10.1073/pnas.0606702104

Fig. 2.

Fig. 2.

Kv1.5 is SUMO modified in vivo and in vitro. (A) Cos-7 cells were cotransfected as described in Materials and Methods with 200 ng of pCDNA3 Ubc9 and, as indicated, 200 ng of pCDNA3 HA-SUMO3, 200 ng of pCDNA3.1 hKv1.5 V5-His, 160 ng of pCDNA3.1 hKv1.5 N556 V5-His, or an equimolar amount of the corresponding empty vector. His-tagged proteins were purified and detected by immunoblotting with anti-HA (Top) and anti-V5 (Middle and Bottom) antibodies. Middle is an overexposed version of Bottom. SUMO-modified species of Kv1.5 are indicated with arrows, and nonspecific bands are indicated with asterisks. (B) Samples were transfected and processed as in A except cells were harvested in the presence or absence of NEM as indicated, and samples were treated with GST or the SUMO-specific protease GST-Ulp1. Arrows indicate SUMO-modified species of Kv1.5. (C) Purified hKv1.5 was incubated with the indicated SUMO conjugation machinery components as described in Materials and Methods. Arrows indicate SUMO-modified species of Kv1.5.