Figure 4
Figure 4. Rap1GAP2, Slp1, and Rab27 form a trimeric complex in vivo. (A) Pull-down of endogenous Rab27 with GST-Slp1 from human platelets. Human platelet lysate was subjected to GST-Slp1 pull-down assay followed by immunoblot analysis using anti-Rab27 antibody, which recognizes both isoforms, Rab27a and Rab27b. (Top panel) Precipitation results. (Bottom panel) Expression levels of endogenous Rab27 protein (total Rab27, 2% input). (B) Coimmunoprecipitation of transfected Rap1GAP2 in complex with Slp1 and Rab27a. HeLa cells were transfected either with VSV-tagged Rab27a alone, together with FLAG-tagged Rap1GAP2, or with FLAG-tagged Rap1GAP2 and myc-tagged Slp1. At 24 hours after transfection, cells were lysed, and Rap1GAP2 was immunoprecipitated using anti-FLAG antibody. The precipitates were analyzed for the presence of Rab27a using anti-VSV antibody. (Top panel) Precipitation results. *Immunoglobulin heavy and light chains. (Bottom 3 panels) Expression levels of transfected Rab27a-VSV, Slp1-myc, and Rap1GAP2-FLAG, each as 2% input. (C) Coimmunoprecipitation of endogenous Rap1GAP2 in complex with Slp1 and Rab27 from human platelets. Human platelet lysate containing endogenous Rab27, Rap1GAP2, and Slp1 was subjected to coimmunoprecipitation using anti-Rab27a antibody. (First and second panels from top) The precipitates were examined for the presence of Rap1GAP2 and Slp1 by immunoblot using specific anti-Rap1GAP2 and anti-Slp1 antibodies. (Third panel from top) Amounts of precipitated Rab27 were controlled with anti-Rab27a antibody. As indicated in panel A, the Rab27 band probably consists of both isoforms, Rab27a and Rab27b. Arrowhead represents precipitated Rab27. *Immunoglobulin light chain. (Bottom 3 panels) Expression levels of endogenous Rap1GAP2 (total RG2), Slp1 (total Slp1), and Rab27 (total Rab27), each as 2% input. Vertical lines have been inserted to indicate a repositioned gel lane in the first and second panels from top as well as in the bottom panel. (D) Colocalization of transfected Rap1GAP2, Slp1, and Rab27a. Colocalization of EGFP-tagged Rab27a, VSV-tagged Rap1GAP2, and myc-tagged Slp1 overexpressed in HeLa cells was analyzed by immunofluorescence as described in “Confocal microscopy.” Arrows represent colocalization of all 3 proteins.

Rap1GAP2, Slp1, and Rab27 form a trimeric complex in vivo. (A) Pull-down of endogenous Rab27 with GST-Slp1 from human platelets. Human platelet lysate was subjected to GST-Slp1 pull-down assay followed by immunoblot analysis using anti-Rab27 antibody, which recognizes both isoforms, Rab27a and Rab27b. (Top panel) Precipitation results. (Bottom panel) Expression levels of endogenous Rab27 protein (total Rab27, 2% input). (B) Coimmunoprecipitation of transfected Rap1GAP2 in complex with Slp1 and Rab27a. HeLa cells were transfected either with VSV-tagged Rab27a alone, together with FLAG-tagged Rap1GAP2, or with FLAG-tagged Rap1GAP2 and myc-tagged Slp1. At 24 hours after transfection, cells were lysed, and Rap1GAP2 was immunoprecipitated using anti-FLAG antibody. The precipitates were analyzed for the presence of Rab27a using anti-VSV antibody. (Top panel) Precipitation results. *Immunoglobulin heavy and light chains. (Bottom 3 panels) Expression levels of transfected Rab27a-VSV, Slp1-myc, and Rap1GAP2-FLAG, each as 2% input. (C) Coimmunoprecipitation of endogenous Rap1GAP2 in complex with Slp1 and Rab27 from human platelets. Human platelet lysate containing endogenous Rab27, Rap1GAP2, and Slp1 was subjected to coimmunoprecipitation using anti-Rab27a antibody. (First and second panels from top) The precipitates were examined for the presence of Rap1GAP2 and Slp1 by immunoblot using specific anti-Rap1GAP2 and anti-Slp1 antibodies. (Third panel from top) Amounts of precipitated Rab27 were controlled with anti-Rab27a antibody. As indicated in panel A, the Rab27 band probably consists of both isoforms, Rab27a and Rab27b. Arrowhead represents precipitated Rab27. *Immunoglobulin light chain. (Bottom 3 panels) Expression levels of endogenous Rap1GAP2 (total RG2), Slp1 (total Slp1), and Rab27 (total Rab27), each as 2% input. Vertical lines have been inserted to indicate a repositioned gel lane in the first and second panels from top as well as in the bottom panel. (D) Colocalization of transfected Rap1GAP2, Slp1, and Rab27a. Colocalization of EGFP-tagged Rab27a, VSV-tagged Rap1GAP2, and myc-tagged Slp1 overexpressed in HeLa cells was analyzed by immunofluorescence as described in “Confocal microscopy.” Arrows represent colocalization of all 3 proteins.

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