R-Ras associates with FLNa at repeat 3.

<p><b>A</b>) Yeast two hybrid screen of R-Ras binding to FLNa. Colonies from + histidine (+his) plates obtained by co-transformation of the L40 yeast strain with BTM116R-Ras-CAAX^- or negative control BTM116-lamin C and VP16-FLNa (nucleotides 1699–1995). The plate was photographed after 3 days of growth. <b>B</b>) R-Ras binds FLNa. GST fusion proteins derived from indicated FLNa fragments were incubated with M2 (FLNa-) cell total lysates. Bound R-Ras was detected by Western blotting for R-Ras. Results are representative of 3 independent experiments. Coomassie stained gel containing purified FLNa fragments is shown to confirm FLNa fragment protein expression. <b>C</b>) R-Ras and FLNa co-immunoprecipitate in M2 melanoma cells. M2 cells were co-transfected with expression vectors for active R-Ras G38V, His-tagged wild type FLNa, His-tagged FLNaΔ3 (deletion of FLNa repeat 3) or vector alone. 48 h post transfection cells were lysed, immunoprecipitated (IP) with anti-His conjugated IgG beads or IgG beads alone and immunoprecipitates were analyzed by immunoblotting with an anti-R-Ras antibody. Input R-Ras and FLNa levels are shown (Bottom). Results are representative of 3 independent experiments. <b>D</b>) The domain structure of FLNa and the location of R-Ras binding site as determined by yeast two hybrid screen. The β-integrin subunits, the actin-binding domain, and the dimerization domain at the C-terminus have been established previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011269#pone.0011269-Pfaff1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011269#pone.0011269-Gorlin1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011269#pone.0011269-Loo1" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011269#pone.0011269-Sharma1" target="_blank">[51]</a>. FLNa fragments repeats 1–10, 11–16, and 17–23 + dimerization domain are shown on the left.