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Conjugation of Rub1 to Cdc53 in vitro depends on Rbx1. Total cell lyaste (200 μg) from Sf21 cells infected with the indicated baculoviruses was immunoprecipitated with 2 μg of anti-Cdc53(yN-18) antibody. Immunoprecipitates were assayed for Rub1 conjugation activity at 25°C for 30 min, and reaction products were analyzed by immunoblotting with anti-Cdc53(yC-17), anti-GST, or anti-myc antibodies.
