Figure 5.

Purified Vph1NT binds to WT Rav1 679–898 in vitro, but not in the presence of the rav1 6Δ mutation. Vph1NT, WT Rav1 679–898-His₆, and Rav1 679–898-His₆ containing the rav1 6Δ mutation were expressed and purified as described under “Experimental procedures.” Vph1NT was mixed with the WT or mutant Rav1 fragment at a 5:1 molar ratio, and the mixture was added to 300 μl of TALON resin and incubated at 4 °C for 2 h. The resin and protein fragments were transferred to a column and washed with low-imidazole buffer, and bound protein was eluted at high imidazole concentrations. 500-μl fractions (F1–F6) were collected for each sample as described under “Experimental procedures.” Input (I), flow-through (FT), wash (W), and fractions are shown for Vph1NT and WT Rav1 679–898-His₆ (Rav1 WT) (A), Vph1NT and Rav1 679–898-His₆ containing the 6Δ mutation (Rav1 6Δ) (B), and Vph1NT only (no His-tagged Rav1) (C). For A and B, a single blot that was cut and the upper portion were probed for Vph1NT (10D7 mouse mAb), whereas the lower portion was probed with anti-His₆.