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. 2007 Sep 4;27(21):7631–7640. doi: 10.1128/MCB.00543-07

FIG. 3.

FIG. 3.

CHC-Tom1L1-Src ternary complex formation. (A) Src-CHC-Tom1L1 ternary complex formation in vitro. Association of indicated purified proteins with Gst-CHC-TD bound to beads. The presence of Src, phosphorylated Src, and phosphorylated Tom1L1 was shown by Western blotting (wb) with the indicated antibodies (αcst1). Association of Gst-CHC-TD with Tom1L1 was revealed by Coomassie brilliant blue staining of the complex separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. (B) Src-CHC-Tom1L1 complex formation in HEK 293 cells that coexpress Src and Tom1L1. Each member of the complex was immunoprecipitated with the indicated antibodies (anti-CHC [α-CHC], anti-Tom1L1 [αTom1L1], and [αcst1]), and the presence of coassociated protein was detected by Western blotting with the antibodies shown. The tyrosine phosphorylation content of each immunoprecipitate is shown. Levels of tyrosine-phosphorylated proteins, CHC, SFK, and Tom1L1 from a whole-cell lysate (WCL) are also shown. (C) Endogenous Tom1L1 bridges SFK to CHC in NIH 3T3 cells. The CHC level associated with SFK was assessed by Western blotting with the indicated antibody after immunoprecipitation of SFK from NIH 3T3 cells that were transfected with the indicated siRNA. The levels of SFK, Tom1L1 and tubulin are also shown. Quantification of associated CHC is indicated. αtubulin, antitubulin antibody. (D) Colocalization of Src, Tom1L1, and CHC. Representative fluorescences of CHC-DsRed, GFP-Tom1L1, and immunostained avian Src are shown with the merged image of an NIH 3T3 cell coexpressing all three components after deconvolution, as described in Materials and Methods. (E) SFK phosphorylates CHC-TD in the presence of Tom1L1. Shown are the results of an in vitro kinase assay using purified Src or Fyn and in the presence of the indicated concentrations of CHC-TD and 1 μM of GST or Gst-Tom1L1, as indicated. Labeled SFK, Gst-Tom1L1, and CHC-TD are shown. (F) Tom1L1 regulates Src-induced CHC phosphorylation in Src-transformed cells. CHC was immunoprecipitated from NIH 3T3 cells or NIH3 3T3 cells stably expressing SrcY527F (Src 527) as shown and that were transfected with control or siRNA Tom1L1 as indicated. The levels of CHC and tyrosine-phosphorylated CHC (pY-CHC) are shown and were assessed by Western blotting with the indicated antibodies. The levels of Tom1L1 and tubulin from indicated cell lysates (WCL) are also shown.