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. 2007 Feb;18(2):501–511. doi: 10.1091/mbc.E06-06-0567

Figure 3.

Figure 3.

Activated ARF6 binds directly to the GDP-bound form of Rac1. (A) Equal amounts of GST, GST-Rac1(ΔCAAX), GST-RhoA, or GST-Cdc42 fusion proteins (Coomassie staining) were incubated with purified ARF6. Using a GST pulldown assay, interacting ARF6 was precipitated and detected by Western blot analysis using a specific anti-ARF6 antibody. These results are representative of three independent experiments, and total input represents 6% of the total protein present in the sample. (B) The GST-Rac1(ΔCAAX) fusion protein coupled to glutathione-Sepharose 4B beads was preloaded with either GDPβS or GTPγS. The nucleotide-bound proteins were incubated with purified ARF6. Interacting proteins were precipitated by a GST pulldown assay, and amounts of associated ARF6 were detected by Western blot analysis using a specific anti-ARF6 antibody. These results are representative of three independent experiments, and the total input represents 6% of the total protein present in the sample. (C) GST-Rac1(ΔCAAX) coupled to glutathione-Sepharose 4B beads was incubated with GDPβS- or GTPγ S-bound purified ARF6. The GST-Rac1 was precipitated and interacting ARF6-GDPβS or ARF6-GTPγS detected by Western blot analysis. These results are representative of four independent experiments. Total input represents 6% of the total protein present in the sample. (D) GST-Rac1(ΔCAAX) coupled to glutathione-Sepharose 4B beads was incubated with GDPβS- or GTPγ S-bound purified ARF1. The GST-Rac1 was precipitated and interacting ARF1-GDPβS or ARF1-GTPγS was detected by Western blot analysis. These results are representative of six independent experiments. Total input represents 30% of the total protein present in the sample. (E) HEK 293 cells stably expressing AT1R-Flag were transfected with Rac1-myc or Rac1T17N-myc and either ARF6-HA, ARF6T157A-HA or empty vector. Using an anti-HA antibody coupled to agarose beads, HA-tagged proteins were immunoprecipitated from the cell lysates, and interacting Rac1 (wild type and mutants) was detected by Western blot analysis using an anti-myc antibody. These results are representative of five independent experiments, and the total input represents 4% of the total protein present in the sample.