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. 2011 Oct 12;31(41):14463–14480. doi: 10.1523/JNEUROSCI.3018-11.2011

Figure 3.

Figure 3.

Characterization of protein regions required for Sytx1A/DCC interaction. A, Diagram summarizing Sytx1A domains and the truncated Sytx1AEGFP chimeras generated. B, Expression of the several Sytx1AEGFP DNAs in HEK293 cells, showing that they generate proteins of the appropriate Mr between 65 and 27 kDa, as revealed by immunoblotting with anti-GFP antibodies. C, Coimmunoprecipitation experiments in HEK293 cells cotransfected with the distinct Sytx1AEGFP constructs together with pCMV or pCMVDCC. DCC immunoprecipitations (200 μg) were revealed by immunoblotting with anti-DCC or anti-GFP antibodies (left panel). Cells cotransfected with pCMVDCC and Sytx1AFLEGFP or Sytx1AH3TMEGFP show positive coimmunoprecipitation of Sytx1A fusions. The reverse immunoprecipitation assays with anti-DCC antibodies also show coimmunoprecipitation with DCC, exclusively when cells are cotransfected with Sytx1AFLEGFP or Sytx1AH3TMEGFP DNAs (arrows in top right panel). The bands corresponding to the distinct Sytx1AEGFP chimeras are labeled with asterisks in the bottom right panel label. D, Diagram summarizing the DCC domains and the truncated EGFPDCC chimeras generated. E, Western blot, revealed with an anti-GFP antibody, demonstrating appropriate Mr (between 90 and 40 kDa) of the distinct EGFPDCC chimeras expressed in HEK293 cells. F, Coimmunoprecipitation experiments in HEK293 cells cotransfected with the distinct EGFPDCC constructs together with pRcCMV or pRcCMVSytx1A DNAs. Sytx1 immunoprecipitations (200 μg) were revealed by immunoblotting with anti-GFP or anti-Sytx1 antibodies (left panel). All the cells cotransfected with pRcCMVSytx1A and the EGFPDCC constructs show coimmunoprecipitation of EGFP-tagged DCC chimeras (arrows), except when cells are cotransfected with EGFPP1-P3DCC DNA. The reverse immunoprecipitation assays with anti-GFP antibodies also reveal coimmunoprecipitation with Sytx1A (arrows in bottom right panel) in all cotransfected cells, except in those transfected with EGFPP1-P3DCC DNA. The bands corresponding to the distinct Sytx1AEGFP chimeras are labeled with asterisks in the top right panel.