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. 2003 Nov 17;22(22):6161–6173. doi: 10.1093/emboj/cdg586

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Fig. 6. The pro-invasive 13mer peptide causes FAK dephosphorylation and δOR serine phosphorylation. (A) HCT-8/E11 cells were left untreated or treated with the 13mer peptide (10–7 M) for 15, 30 or 60 min, followed by solubilization in lysis buffer. Cell lysates were immunoprecipitated with an antibody against tyrosine-phosphorylated residues (PY-20). The immunoprecipitates were immunoblotted with anti-FAK (upper panel) or anti-v-Src (lower panel; lc, loading control) antibodies. Immunoblots were quantified as in Figure 4B (right panel). *Significantly different from untreated at p < 0.005. (B) HCT-8/E11 cells were treated as in (A). Cell lysates were immunoprecipitated with an anti-v-Src antibody. The immunoprecipitates were immunoblotted with an anti-FAK antibody (upper panel), a recombinant antibody, specific for tyrosine-phosphorylated residues (RC20-HRP) (middle panel) or an anti-v-Src antibody (lower panel; lc, loading control). Immunoblots were quantified as in Figure 4B (right panel). (C) HCT-8/E11 cells were left untreated (–) or treated for 60 min (+) with the 13mer peptide (10–7 M), Y27632 (10–6 M), C3 exoenzyme (6.6 µg/ml) or LY294002 (10–5 M), followed by solubilization in lysis buffer. Immunoprecipitates were prepared and probed as in (A) (upper panel). A non-identified tyrosine phosphorylated protein crossreacting with the anti-FAK antibody is presented as loading control (lc). Immunoblots were quantified as in Figure 4B (right panel). *Significantly different from untreated (–) cells at p < 0.005. (D) HCT-8/E11 cells were left untreated (–) or treated (+) for 5, 15 or 30 min with the 13mer peptide (10–7 M) and/or with naloxone (10–5 M). Cell lysates were immunoprecipitated using an anti-phosphoserine antibody and immunoblotted with an anti-δOR antibody. A non-identified serine phosphorylated protein, crossreacting with the anti-δOR antibody, is presented as loading control (lc). Immunoblots were quantified as in Figure 4B (right panel). *Significantly different from untreated (–) cells at p < 0.005.