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. 2002 May 21;99(11):7450–7454. doi: 10.1073/pnas.112198799

Figure 1.

Figure 1

(A) Purified TIP47 mutant proteins. Twelve mutants were generated that contain three alanines in place of residues 152–187 as described in Methods. Proteins were expressed in E. coli and purified. Shown is a 12.5% SDS-polyacrylamide gel stained with Coomassie blue. Lane 1, TIP47 wild type. Lanes 2–12, TIP47 mutants 1–10 and 12. TIP47 mutant 11 is not shown. The specific three residues mutated in each mutant protein are listed in B. (B) Alanine scanning mutagenesis reveals specific residues in TIP47 important for Rab9 binding. His-tagged TIP47 mutants (200 nM) were assayed for Rab9 (200 nM) binding. The data were corrected for background binding obtained by using His-tagged GFP instead of His-TIP47 and are representative of at least two, and for most mutants three, independent experiments. The dashed line is included to aid comparison with the wild-type binding level observed. Shown below, anti-Rab9 immunoblot data used for this analysis.