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. 1999 Nov 15;13(22):2928–2933. doi: 10.1101/gad.13.22.2928

Figure 4.

Figure 4

Figure 4

Conjugation of Rub1 to Cul2 depends on Rbx1. (A) Cell lysate (20 μg) from the indicated baculovirus-infected Sf21 cells was immunoblotted with anti-HA or anti-myc antibodies. Total cell lyaste protein (200 μg) from baculovirus-infected Sf21 cells was immunoprecipitated with 2 μg of anti-myc antibody. The beads were incubated with 30 ng of Ula1, 20 ng of Uba3, 100 ng of Ubc12, 1 μg of GST–Rub1, and 1.5 mm ATP at 25°C for 50 min and immunoblotted with ant-HA or anti-GST antibodies. (B) After immunoprecipitation with 12 μg of anti-myc antibody from 1.2 mg of baculovirus infected Sf21 cell lysate, the beads were divided into six tubes and incubated at 25°C for 30 min in the presence or absence of Uba3/Ubc12, GST–Rub1, and ATP as indicated and immunoblotted with anti-HA or anti-GST antibodies.