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Aits. The immunoblots were developed with specific antibodies against the indicated proteins. Glutathione-sepharose beads with whole cell lysate (B + K562), lysis buffer with the corresponding GST-fused proteins (LB) and pGEX-4T-1 empty plasmid with K562 whole cell lysate (GST) were used as negative controls. L: whole cell lysate (40 g). The asterisks indicate unspecific bands.baits. Both Abi1-SH3 and SH3-b domains bound to BcrAbl, in agreement with the literature [31]. In addition, both domains interacted with p87C3G and a slight interaction of the Abi1-SH3-b domain with p140C3G was also detected (Figure 1B). Regarding p130Cas, Figure 1C shows that the three tested domains (SH3-binding, P1 and P2) interact with Bcr-Abl, albeit with different affinities. Additionally, p130Cas-SH3 domain interacted with p140C3G, in agreement with other studies [29], while P1 and P2 domains associate preferentially with p87C3G. On the other hand, Cbl-SH3-b domain clearly interacts with both Bcr-Abl and p87C3G (Figure 1D). Association of Cbl with Bcr-Abl, as well as with CrkL or Grb2 (used as controls) in PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/11834444 K562 cells has been described previously [24], although the interaction described by these authors involved the Bcr-Abl SH2 domain and Cbl phospho-tyrosines. The preferential interaction of p87C3G over p140C3G with most of the tested domains probably reflects its highest expression in CML cells (Additional file 1) [8].These experiments support the results derived from the Bcr-Abl-SH3 domain pull-down assays and suggest that all these proteins form complexes. In fact, all of them coimmunoprecipitate (Additional file 2). Therefore, Cbl, Abi1 and p130Cas are clear candidates to act as intermediates in the interaction between Bcr-Abl and C3G in K562 cells. In agreement with pull-down assays, confocal microscopy revealed a colocalization of C3G with phospho-p130Cas, Cbl, Abi1 and Bcr-Abl (Figure 2), which further supports that all these proteins form complexes in K562 cells.Interaction of Bcr-Abl with CrkLCrkL, the major substrate of the tyrosine kinase Bcr-Abl, interacts directly with Bcr-Abl through its amino terminal SH3 domain and the SH3-binding region of Abl [36,37]. Here we show that the Abl-SH3 domain also interacts with CrkL (Additional file 3A). One possible explanation is that CrkL, similarly to CrkII, (R)-1-(3-Chlorophenyl)ethan-1-ol has a putative SH3-b domain inside the SH2 domain that couldMaia et al. Cell Communication and Signaling 4-Bromo-5-fluoro-2-nitrophenol 2013, 11:9 http://www.biosignaling.com/content/11/1/Page 4 ofDAPIC3G-FITCp-p130Cas-CyMergedDICCTC3G-FITCCbl-CyC3G-FITCAbi1-CyC3G-FITCAbl-CyFigure 2 C3G colocalizes with phospho-p130Cas, Cbl, Abi1 and Bcr-Abl in K562 cells. Confocal microscopy images of K562 cells adhered to fibronectin covered slides (10 g/ml) and labeled with anti-C3G (G4, mouse monoclonal, rows 1 and 2 or 1008, rabbit polyclonal, rows 3 and 4) and either, anti-phospho-p130Cas (rabbit polyclonal), anti-Cbl (rabbit polyclonal), anti-Abi1 (mouse monoclonal) or anti-Abl (mouse monoclonal) specific antibodies as indicated. Anti-FITC and anti-Cy3 were used as secondary antibodies. Nuclei were labeled with DAPI. Control cells (CT) were incubated with DAPI plus the secondary antibodies. DIC: Differential interference contrast microscopy. p-p130Cas: phospho-p130Cas. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8833965 The bars represent 10 m.mediate this interaction [38]. Sequence alignment revealed that CrkL lacks the SH3-b domain present in CrkII, although it preserves a canonical SH3-b PXXP motif that itself is sufficient for binding to.