Figure 3
Binding of Rap1GAP2 to Slp1 is mediated through the TKXT motif within the C-terminus of Rap1GAP2. (A) Pull-down of transfected Rap1GAP2 truncation mutants with GST-Slp1. HeLa cells were transfected with either FLAG-tagged Rap1GAP2 (RG2-FLAG) wild-type or different Rap1GAP2 truncation mutants. Rap1GAP2ΔCterm lacks amino acids 467-715, whereas Rap1GAP2ΔNterm lacks amino acids 1-121. Cells were lysed, and pull-down assays using GST or GST-Slp1 were performed. Precipitates were analyzed for the presence of FLAG-tag containing proteins by immunoblot with anti-FLAG antibody. (Top panel) Precipitation results. (Bottom panel) Expression levels of RG2 proteins (total RG2-FLAG, 2% input). (B) Pull-down of transfected Rap1GAP2 deletion mutants with GST-Slp1. Lysates of HeLa cells overexpressing either FLAG-tagged Rap1GAP2 wild-type (RG2-FLAGwt) or deletion mutants Rap1GAP2Δ536-542 as control and Rap1GAP2Δ522-527 were subjected to GST-Slp1 pull-down assays followed by immunoblot analysis using anti-FLAG antibody. (Top panel) Precipitation results. (Bottom panel) Expression levels of RG2 proteins (total RG2-FLAG, 2% input). (C) Pull-down of transfected Rap1GAP2 alanine point mutants with GST-Slp1. HeLa cells were transfected with FLAG-tagged Rap1GAP2 wild-type (RG2-FLAGwt) or different Rap1GAP2 point mutants having each of the amino acids within the EVTKTT sequence (amino acids 522-527) changed to alanine as indicated. Cells were lysed, and lysates were subjected to GST-Slp1 pull-down assays followed by immunoblot analysis with anti-FLAG antibody. (Top panel) Precipitation results. (Bottom panel) Expression levels of RG2 proteins (total RG2-FLAG, 2% input). (D) Peptide binding assay (PepSpot). Synthetic Rap1GAP2 (RG2) peptides covalently bound to cellulose membrane containing either wild-type EVTKTT sequence of Rap1GAP2 or with consecutive amino acid changed to alanine (A) or phosphorylated threonine residues (pT) as indicated were subjected to GST-Slp1 overlay assay followed by immunoblot analysis using anti-GST antibody. (E) Pull-down of endogenous Rap1GAP2 with GST-Slp1 from human platelet lysates in absence or presence of Rap1GAP2 peptides. Human platelet lysate or lysate supplemented with Rap1GAP2 wild-type (RG2wt) peptide (amino acids 518-532 of Rap1GAP2) or Rap1GAP2ΔEVTKTT (RG2ΔEVTKTT) peptide (amino acids 515-535 of Rap1GAP2 lacking amino acids 522-527) in dimethyl sulfoxide 100 μM each were subjected to GST-Slp1 pull-down assays. The presence of endogenous Rap1GAP2 protein (RG2) was analyzed by immunoblot using anti-Rap1GAP2 antibody. (Top panel) Precipitation results. (Bottom panel) Expression levels of endogenous Rap1GAP2 protein (total RG2, 2% input).

Binding of Rap1GAP2 to Slp1 is mediated through the TKXT motif within the C-terminus of Rap1GAP2. (A) Pull-down of transfected Rap1GAP2 truncation mutants with GST-Slp1. HeLa cells were transfected with either FLAG-tagged Rap1GAP2 (RG2-FLAG) wild-type or different Rap1GAP2 truncation mutants. Rap1GAP2ΔCterm lacks amino acids 467-715, whereas Rap1GAP2ΔNterm lacks amino acids 1-121. Cells were lysed, and pull-down assays using GST or GST-Slp1 were performed. Precipitates were analyzed for the presence of FLAG-tag containing proteins by immunoblot with anti-FLAG antibody. (Top panel) Precipitation results. (Bottom panel) Expression levels of RG2 proteins (total RG2-FLAG, 2% input). (B) Pull-down of transfected Rap1GAP2 deletion mutants with GST-Slp1. Lysates of HeLa cells overexpressing either FLAG-tagged Rap1GAP2 wild-type (RG2-FLAGwt) or deletion mutants Rap1GAP2Δ536-542 as control and Rap1GAP2Δ522-527 were subjected to GST-Slp1 pull-down assays followed by immunoblot analysis using anti-FLAG antibody. (Top panel) Precipitation results. (Bottom panel) Expression levels of RG2 proteins (total RG2-FLAG, 2% input). (C) Pull-down of transfected Rap1GAP2 alanine point mutants with GST-Slp1. HeLa cells were transfected with FLAG-tagged Rap1GAP2 wild-type (RG2-FLAGwt) or different Rap1GAP2 point mutants having each of the amino acids within the EVTKTT sequence (amino acids 522-527) changed to alanine as indicated. Cells were lysed, and lysates were subjected to GST-Slp1 pull-down assays followed by immunoblot analysis with anti-FLAG antibody. (Top panel) Precipitation results. (Bottom panel) Expression levels of RG2 proteins (total RG2-FLAG, 2% input). (D) Peptide binding assay (PepSpot). Synthetic Rap1GAP2 (RG2) peptides covalently bound to cellulose membrane containing either wild-type EVTKTT sequence of Rap1GAP2 or with consecutive amino acid changed to alanine (A) or phosphorylated threonine residues (pT) as indicated were subjected to GST-Slp1 overlay assay followed by immunoblot analysis using anti-GST antibody. (E) Pull-down of endogenous Rap1GAP2 with GST-Slp1 from human platelet lysates in absence or presence of Rap1GAP2 peptides. Human platelet lysate or lysate supplemented with Rap1GAP2 wild-type (RG2wt) peptide (amino acids 518-532 of Rap1GAP2) or Rap1GAP2ΔEVTKTT (RG2ΔEVTKTT) peptide (amino acids 515-535 of Rap1GAP2 lacking amino acids 522-527) in dimethyl sulfoxide 100 μM each were subjected to GST-Slp1 pull-down assays. The presence of endogenous Rap1GAP2 protein (RG2) was analyzed by immunoblot using anti-Rap1GAP2 antibody. (Top panel) Precipitation results. (Bottom panel) Expression levels of endogenous Rap1GAP2 protein (total RG2, 2% input).

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