Figure 1.
Figure 1. In vitro assays of protein activity. (A and B) Guanosine nucleotide exchange assays. (A) Release of [3H]GDP from 150 nM Tem1 without (open circles) or with 790 nM Lte1 (closed circles). (B) Release of [3H]GDP from a Tem1–Bfa1 complex (150 nM/340 nM) was assayed in the presence of 0 (open circles), 420 (triangles), or 840 nM (closed circles) Lte1. (C) Bfa1 inhibits intrinsic nucleotide exchange of Tem1. Increasing concentrations of Bfa1 were added to 1.8 µM Tem1. The percentage of GDP bound is the amount of nucleotide remaining bound to Tem1 after a 4-min incubation at 25°C. (D) Lte1–Ras2V19 interaction. Binding of 6His-Lte1 to glutathione beads carrying GST or GST-Ras2V19 was assayed in a Western blot (WB) using anti-6His antibody. Input amounts of GST and GST-Ras2V19 are visualized by Coomassie blue staining. Molecular mass is indicated in kilodaltons.

In vitro assays of protein activity. (A and B) Guanosine nucleotide exchange assays. (A) Release of [3H]GDP from 150 nM Tem1 without (open circles) or with 790 nM Lte1 (closed circles). (B) Release of [3H]GDP from a Tem1–Bfa1 complex (150 nM/340 nM) was assayed in the presence of 0 (open circles), 420 (triangles), or 840 nM (closed circles) Lte1. (C) Bfa1 inhibits intrinsic nucleotide exchange of Tem1. Increasing concentrations of Bfa1 were added to 1.8 µM Tem1. The percentage of GDP bound is the amount of nucleotide remaining bound to Tem1 after a 4-min incubation at 25°C. (D) Lte1–Ras2V19 interaction. Binding of 6His-Lte1 to glutathione beads carrying GST or GST-Ras2V19 was assayed in a Western blot (WB) using anti-6His antibody. Input amounts of GST and GST-Ras2V19 are visualized by Coomassie blue staining. Molecular mass is indicated in kilodaltons.

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