Fig. 2. Isolation of MBF1-associated proteins. (A) FLAG-MBF1-associated proteins. Proteins from nuclear extracts of the FLAG-MBF1-expressing transgenic line F (lane 2) were captured on an anti-FLAG affinity column, separated by 10% SDS-PAGE and silver stained. Nuclear extracts of the parental yw line (lane 1) served as a control. Positions of molecular weight markers are shown on the left. (B) Western analyses of the captured proteins. The same samples as in A were resolved on SDS-PAGE, blotted onto membranes and probed with the indicated antibodies. (C) GST pull-down assay for interaction between MBF1 and TDF. Bacterially expressed and purified His-MBF1 (100 ng) was incubated with 200 ng of either GST (lane 2), GST-TDF (lane 3) or the GST-TDF-C56 fusion with the N-terminally truncated TDF (lane 4). The bound His-MBF1 was resolved by 15% SDS-PAGE and detected with the anti-MBF1 antibody. Lane 1 contains 1/10 of input His-MBF1. (D) GST pull-down assay for interaction between MBF1 and TBP. Bacterially expressed and purified His-TBP (100 ng) was incubated with 200 ng of either GST (lane 2) or GST-MBF1 (lane 3). The bound TBP was resolved by 8% SDS-PAGE and detected with the anti-TBP antibody. Lane 1 contains 1/10 of input His-TBP.