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. 2009 Jul 1;106(29):12037–12042. doi: 10.1073/pnas.0903869106

Fig. 1.

Fig. 1.

Analysis of the Nej1-Srs2 interaction. (A) Shown is the 2-hybrid strain PJ69–4A containing pAS1-NEJ1 (Gal4BD-NEJ1) or pASI-nej1 substitution derivatives as bait. Prey constructs contain the indicated LIF1 or SRS2 gene fusions in pACTII (Gal4AD). Cells are spotted as 10-fold serial dilutions on 2-hybrid indicator medium lacking adenine or histidine. (B) Two-hybrid analysis using the indicated bait and prey constructs in a dun1 derivative of PJ69–4A. (C) Left, Radiograph of an MBP pull-down is shown. Lanes show input, pull-downs with MBP alone and pull-downs with MBP-Nej1, as indicated. The in vitro translated protein added was Lif1 (lanes 1–3), full-length Srs2 (lanes 4–6), and amino acids 810 to 1,174 of Srs2 (lanes 7–9). Arrows indicate the recovery of full-length Srs2 and amino acids 810 to 1,174 of Srs2. Higher mobility peptides in the input and pull-down lanes are interpreted to represent truncated forms of the respective in vitro translated proteins. Size markers are shown to the left. Right, Coomassie staining of the same gel used to generate the autoradiograph is shown.