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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.
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