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Fig. 5 | Cell Communication and Signaling

Fig. 5

From: The phosphatase Shp1 interacts with and dephosphorylates cortactin to inhibit invadopodia function

Fig. 5

GroPIns favors the association between Shp1 and cortactin. A Representative pull-down assay of His-Shp1 with Flag-cortactin-beads from A375MM lysates over-expressing Flag-cortactin. Beads were treated with buffer alone (−) or with GroPIns and GroPIns4P (as indicated), and then incubated with purified His-Shp1. The eluted and unbound proteins were analyzed by western blotting using an anti-Shp1 antibody, while immunopurified Flag-cortactin was revealed using an anti-cortactin antibody. Shp1 staining is shown with two different exposure times (short and long) for better interpretation. B Representative in vitro pull-down assay of cortactin, previously phosphorylated on tyrosine residues by Src kinase in an in vitro kinase assay, with His-Shp1. The unbound and eluted proteins were analyzed by western blotting using anti-phosphotyrosine and anti-cortactin antibodies. His-Shp1 was revealed by Ponceau staining. C, D Interaction between Shp1 C455S mutant and cortactin was examined by immunoprecipitation (IP) with an anti-Flag (C) or anti-Shp1 (D) antibodies in A375MM cells over-expressing Shp1 C455S mutant and Flag-cortactin, untreated (−) or treated (+) with 50 μM GroPIns for 30 min (as indicated). Representative western blotting with anti-Shp1 and anti-cortactin antibodies, of total lysate (input), unbound and immunoprecipitated proteins (as indicated). Molecular weight standards (kDa) are indicated on the left of each panel. The blots shown are representative of at least two different experiments

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