mutants have a biochemical defect in Rub1p removal from Cdc53p
(A) Total cell lysate was prepared from CSN containing Δrub1 and csn deficient Δrri1/csn5 strains according to the protocol described in Fig. 1C. Individual lysates (lanes 1–12) and a 1: 2 mixture of the csn
Δrub1 and Δrri1/csn5 lysates (lanes 13–18) were incubated for the indicated times (0–60 minutes) at 30°C, followed by immunoblotting with Cdc53p antibodies. Unmodified and Rub1p-modified Cdc53p are indicated. (B) Total cell lysate of the indicated csn mutants was prepared as described in Fig. 1B and incubated with 150 ng of purified human CSN complex for 2h at 30°C. The effect on Cdc53p modification was assessed by immunoblotting as described in Fig. 1C. Unmodified and Rub1p-modified Cdc53p are indicated. (C) Coomassie blue stained gel showing the purified human CSN preparation used in the experiments in Fig. 2B. Human CSN was purified as described , and individual subunits as determined by immunoblotting and mass spectrometry are indicated.