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. 2009 Mar 23;184(6):785–792. doi: 10.1083/jcb.200810155

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© 2009 Zhang et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — RIL inhibits Src activation and reduces anchorage-independent growth. (A) Lysates (15 µg/lane) of WI-38 (lane 1), HCT116 (lane 2), HT29 (lane 3), RKO (lane 4), Caco-2 (lane 5), DLD-1 (lane 6), SK-LMS-1 (lane 7), HeLa (lane 8), and 293 (lane 9) cells were analyzed by Western blotting with antibodies recognizing RIL or tubulin (as a loading control). (B) HCT116 cells were transfected with Flag-RIL vector (lane 2) or a vector lacking the RIL sequence as a control (lane 1). The lysates were probed with antibodies recognizing RIL, Src, pY419 Src, or pY530 Src. (C) Src was immunoprecipitated from HCT116 cells transfected with the control (lane 1) or Flag-RIL (lane 2) vector and analyzed by Western blotting with an anti-Src antibody. (D) The kinase activity of Src derived from the Flag-RIL transfectants was compared with that of the control cells. (E) FAK was immunoprecipitated from HCT116 cells transfected with the control (lane 1) or Flag-RIL (lane 2) vector and analyzed by Western blotting with antibodies specific for FAK or pY566/577 FAK. IP, immunoprecipitation. (F) Anchorage-independent growth of HCT116 cells in the presence of PP2 or PP3 was compared with that of HCT116 cells grown in the absence of PP2 and PP3 (control). (G and H) HCT116 cells were transfected with the control vector (G, lane 1), the Flag-RIL vector (G, lane 2), or the Flag-RIL vector and a vector encoding a constitutively active form of chicken Src (Y527F; G, lane 3). (G) The lysates were probed with antibodies recognizing RIL, pY419 Src, or Src. (H) Anchorage-independent growth of the cells expressing Flag-RIL or Flag-RIL and constitutively active Src was compared with that of the control cells. (D, F, and H) Error bars represent mean ± SD (n = 3). Mr, molecular mass.