Figure 1
Figure 1. Rap1GAP2 interacts with Slp1. (A) Pull-down of transfected Rap1GAP2 with GST-Slp1. COS-1 cells were transfected with FLAG-tagged Rap1GAP2 (RG2-FLAG). (Bottom panel) Equal amounts of GST as control and GST-Slp1 coupled to GSH-Sepharose beads were used for precipitation. (Top panel) Bound Rap1GAP2 protein was visualized by immunoblot using anti-FLAG antibody. Expression level of Rap1GAP2 is shown as 2% input of total RG2-FLAG. The broad band of Rap1GAP2 is probably the result of extensive posttranslational modifications. (B) Coimmunoprecipitation of transfected Rap1GAP2 and Slp1. COS-1 cells were transfected with FLAG-tagged Rap1GAP2, myc-tagged Slp1, or FLAG-tagged Rap1GAP2 together with myc-tagged Slp1. After lysis, Rap1GAP2 was precipitated with anti-FLAG antibody. The precipitates were analyzed for the presence of bound Slp1 by immunoblot using anti-myc antibody. (Top panel) Precipitation results. (Bottom 2 panels) Transfection levels of Rap1GAP2 (total RG2-FLAG, 2% input) and Slp1 (total Slp1-myc, 2% input). (C) Pull-down of endogenous Rap1GAP2 with GST-Slp1 from human platelets. Equal amounts of GST as control and GST-Slp1 coupled to GSH-Sepharose beads were incubated with human platelet lysate. Bound endogenous Rap1GAP2 protein was visualized with anti-Rap1GAP2 antibody (RG2). Expression level of Rap1GAP2 is shown as 2% input of total RG2. (D) Pull-down of transfected Rap1GAP2 and Slp1 with GST-14-3-3. COS-1 cells were transfected with myc-tagged Slp1 and without or with FLAG-tagged Rap1GAP2 (RG2-FLAG). Cells were lysed, and GST-14-3-3β was used to pull down Rap1GAP2 and indirectly Slp1 bound to Rap1GAP2. (Top panel) Precipitated Slp1 was visualized by immunoblotting using anti-myc antibody. (Second panel from top) Precipitation of Rap1GAP2 was controlled by immunoblot with anti-FLAG antibody. In parallel, total cell lysates were analyzed for the expression of Slp1 (total Slp1-myc, 2% input) and Rap1GAP2 (total RG2-FLAG, 2% input). (E) Pull-down of endogenous Rap1GAP2 and Slp1 from human platelets. Lysates from thrombin-treated platelets were subjected to pull-down assays using either GST as control or GST-14-3-3β. (First and second panels from top) The precipitates were analyzed for the presence of endogenous Rap1GAP2 (RG2) and Slp1 using specific anti-Rap1GAP2 and anti-Slp1 antibodies. Expression levels of Rap1GAP2 and Slp1 are shown as 2% input of total protein amounts. (Bottom panel) The amounts of GST and GST-14-3-3β used for precipitation. *Unspecific band.

Rap1GAP2 interacts with Slp1. (A) Pull-down of transfected Rap1GAP2 with GST-Slp1. COS-1 cells were transfected with FLAG-tagged Rap1GAP2 (RG2-FLAG). (Bottom panel) Equal amounts of GST as control and GST-Slp1 coupled to GSH-Sepharose beads were used for precipitation. (Top panel) Bound Rap1GAP2 protein was visualized by immunoblot using anti-FLAG antibody. Expression level of Rap1GAP2 is shown as 2% input of total RG2-FLAG. The broad band of Rap1GAP2 is probably the result of extensive posttranslational modifications. (B) Coimmunoprecipitation of transfected Rap1GAP2 and Slp1. COS-1 cells were transfected with FLAG-tagged Rap1GAP2, myc-tagged Slp1, or FLAG-tagged Rap1GAP2 together with myc-tagged Slp1. After lysis, Rap1GAP2 was precipitated with anti-FLAG antibody. The precipitates were analyzed for the presence of bound Slp1 by immunoblot using anti-myc antibody. (Top panel) Precipitation results. (Bottom 2 panels) Transfection levels of Rap1GAP2 (total RG2-FLAG, 2% input) and Slp1 (total Slp1-myc, 2% input). (C) Pull-down of endogenous Rap1GAP2 with GST-Slp1 from human platelets. Equal amounts of GST as control and GST-Slp1 coupled to GSH-Sepharose beads were incubated with human platelet lysate. Bound endogenous Rap1GAP2 protein was visualized with anti-Rap1GAP2 antibody (RG2). Expression level of Rap1GAP2 is shown as 2% input of total RG2. (D) Pull-down of transfected Rap1GAP2 and Slp1 with GST-14-3-3. COS-1 cells were transfected with myc-tagged Slp1 and without or with FLAG-tagged Rap1GAP2 (RG2-FLAG). Cells were lysed, and GST-14-3-3β was used to pull down Rap1GAP2 and indirectly Slp1 bound to Rap1GAP2. (Top panel) Precipitated Slp1 was visualized by immunoblotting using anti-myc antibody. (Second panel from top) Precipitation of Rap1GAP2 was controlled by immunoblot with anti-FLAG antibody. In parallel, total cell lysates were analyzed for the expression of Slp1 (total Slp1-myc, 2% input) and Rap1GAP2 (total RG2-FLAG, 2% input). (E) Pull-down of endogenous Rap1GAP2 and Slp1 from human platelets. Lysates from thrombin-treated platelets were subjected to pull-down assays using either GST as control or GST-14-3-3β. (First and second panels from top) The precipitates were analyzed for the presence of endogenous Rap1GAP2 (RG2) and Slp1 using specific anti-Rap1GAP2 and anti-Slp1 antibodies. Expression levels of Rap1GAP2 and Slp1 are shown as 2% input of total protein amounts. (Bottom panel) The amounts of GST and GST-14-3-3β used for precipitation. *Unspecific band.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal