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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 3. RGS14 binds Raf kinases.

Figure 3

(A) Protein interaction trap (pull-down) assays to detect Raf complexed with purified RGS14. Cell lysates expressing either HA-Raf-1 or HA-B-Raf were incubated with purified Trx-H6-RGS14 and, following recovery by Ni-NTA, proteins were detected by anti-HA (Raf-1 or B-Raf) or anti-His (RGS14) antibody, respectively. (B) RGS14 co-immunoprecipitates (IP) with Raf kinases from cell lysates when expressed in cells. HeLa cells were co-transfected with Flag-tagged (Flag-RGS14) together with individual HA-tagged Raf kinase (HA-Raf-1 or HA-B-Raf) isoforms, respectively. Whole-cell extracts were subjected to immunoprecipitation followed by immunoblot with the indicated antibodies. The cell lysate shown in the lanes represents 5% of the whole cell extracts used for IP. (C) RGS14 and Raf kinases co-localize in the cytosol of cells. RGS14-GFP and Rafs (HA-Braf or HA-Raf-1) were co-transfected into HeLa cells. After transfection, cells were fixed, and Rafs were immunostained with anti-HA antibody (Raf-1 or B-Raf) as described. RGS14-GFP was detected by autofluorescence. Proteins were visualized by confocal microscopy as described. Scale bars equal 10 µm. Results are representative of at least three separate experiments for each condition.