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. Author manuscript; available in PMC: 2011 Mar 1.
Published in final edited form as: Cell Signal. 2010 Mar;22(3):366–376. doi: 10.1016/j.cellsig.2009.10.005

Figure 9. Giα1 does not bind Raf kinases, but does recruit Raf kinases to the plasma membrane in cells.

Figure 9

(A) Giα1 does not bind Raf kinases. Purified His6-Giα1 was incubated with cell extracts expressing either HA-Raf-1 or HA-B-Raf kinase. Samples were subjected to protein interaction trap assays (Ni-NTA pull-down) and recovered proteins were detected by immunoblot analysis. (B) Giα1 and Raf kinases co-localize at the plasma membrane. EE-tagged Giα1, Raf-1 or B-Raf were transfected into HeLa cells alone or Giα1-EE and each Raf kinase were co-transfected together. After transfection, cells were fixed, and Rafs were detected by immunostaining with anti-HA antibody and Giα1 was detected using the anti-EE antibody, as described. (C) Giα1 recruits RGS14 and Raf kinases to the plasma membrane. HeLa cells were transfected with RGS14-GFP, Giα1-EE and either HA-Raf-1 or HA-B-Raf., respectively. Rafs and Giα1 were detected as described above. RGS14-GFP was detected by autofluorescence of GFP. Proteins were visualized by confocal microscopy as described. Scale bars equal 10µm. Results are representative of at least three separate experiments for each condition.