approved the final version of the manuscript. REFERENCES 1. Finally, direct binding to c-Src only occurred through the PLC- SH2 domains, an conversation that was prevented by blocking the PLC- SH2 domain name. This study exhibited that c-Src and subsequently purified with glutathione agarose beads (Sigma). Full-length His6-tagged human recombinant PLC- purified protein was from Calbiochem. Full-length active human c-Src was from Millipore. BODIPY-conjugated peptides. Peptide synthesis and purification were performed by BIX 01294 the Synthesis and Sequencing Facility at Johns Hopkins University or college School of Medicine. Coupling of the peptide to BODIPY 577/618 maleimide was performed according to the manufacturer’s protocol (Invitrogen). PLC- SH2 domain name binding (i.e., active) and unfavorable control (i.e., inactive) peptides have been previously explained (7, 59). Measurement of Na+/H+ exchange. Cellular Na+/H+ exchange activity in Caco-2/BBe/NHE3 cells produced to 14 days postconfluency on Transwell filters was decided fluorometrically using the intracellular pH-sensitive dye 2,7-bis(carboxyethyl)5C6-carboxyfluoresceinacetoxy-methyl ester (BCECF-AM; 5 M; Molecular Probes, Eugene, OR), as explained previously (28). Caco-2/BBe/NHE3 cells were exposed to 50 mM NH4Cl during a 45-min dye loading, as explained previously (42, 57, 59). Cells were perfused in the beginning with TMA+ answer alone or with 10 M CCh for 1C10 min (130 mM tetramethylammonium chloride, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 25 mM glucose, and 20 mM HEPES, pH 7.4) before being switched to Na+ answer (130 mM NaCl instead of tetramethyl-ammoniumchloride) for the Na+-dependent pHi recovery. At the end of each experiment, the fluorescence ratio was calibrated to pHi using the high potassium/nigericin method. Na+/H+ exchange activity data were calculated as the ratio of Na+-dependent changes in pHi over initial time (pH/min) of Na+-dependent pH recovery using at least three coverslips per condition in a single experiment. Initial rates were analyzed by using Origin (Microcal Software) to determine statistical significance among individual experiments. Means SE were decided from at least three individual experiments. Protein-protein interactions. Protein overlay (Much Western) assays were used to examine the conversation of PLC- purified proteins (2 g of each full-length and -specific array domains) on blots with recombinant c-Src (overlay) by subsequent incubation of blots with monoclonal anti-Src antibody, as explained previously (55). For peptide competition studies, PLC- purified proteins were separated on SDS-PAGE, and proteins were transferred to nitrocellulose membranes and exposed to 40 g of either the active or inactive peptides conjugated to BODIPY 577/618 for 1 h at room heat. Binding of peptides was visualized using a fluorescent Typhoon Imager (Johns Hopkins University or college School of Medicine Proteomics Core). Membranes were then exposed to 4 g purified full-length Src recombinant protein overnight at 4C. c-Src binding was determined by Western blotting as explained above. Results were obtained from three individual experiments. Coimmunoprecipitation. PLC- or NHE3 were immunoprecipitated (IP) from the total lysate of Caco-2/BBe/NHE3 cells (in the presence of 1% Triton X-100). All IPs were carried out at 4C with constant mixing on a rotary shaker. Briefly, each sample (1 mg of total cell lysate per IP) was first precleared with either protein A-Sepharose beads (Sigma) or protein A beads conjugated to BIX 01294 rabbit anti-mouse secondary antibody for 1 h. The precleared lysate was then incubated with 4 g of antibodies to PLC- or c-Src for 1 h. Protein A-Sepharose beads were then added to each IP combination, and incubation was continued for another 1 h. The beads were washed four occasions with phosphate-buffered saline made up of 0.1% Tween 20 (Sigma). The IP pellets were analyzed by SDS-PAGE and Western blotted with corresponding antibodies. Caco-2/BBe cells were produced on 10-cm2 Transwell Petri dishes until postconfluent for 12 days and infected with adenovirus 3HA-NHE3 construct as explained above. After contamination, cells were allowed to recover in normal media for 40 h before CCh treatment. On postconfluency, cells were serum-starved again for 4 h and treated either with vehicle or 10 M CCh (Sigma) for 1C10 min at 37C. Adenovirus infected Caco-2/BBe cells were washed three times in ice-cold phosphate-buffered saline made up of 50 mM Tris. Cells were collected and lysed in 500 l of ice-cold lysis buffer (10 mM HEPES, 50 mM NaCl, 5 mM.We as well as others have demonstrated that several proteins, including NHERF2, bind to the C terminus of NHE3 in the same area (aa 585C605), which has been shown to be necessary for PLC- binding. the PLC- SH2 domain. This study exhibited that c-Src and subsequently purified with glutathione agarose beads (Sigma). Full-length His6-tagged human recombinant PLC- purified protein was from Calbiochem. Full-length active human c-Src was from Millipore. BODIPY-conjugated peptides. Peptide synthesis and purification were performed by the Synthesis and Sequencing Facility at Johns Hopkins University or college School of Medicine. Coupling of the peptide to BODIPY 577/618 maleimide was performed according to the manufacturer’s protocol (Invitrogen). PLC- SH2 domain name binding (i.e., active) and unfavorable control (i.e., inactive) peptides have been previously explained (7, 59). Measurement of Na+/H+ exchange. Cellular Na+/H+ exchange activity in Caco-2/BBe/NHE3 cells grown to 14 days postconfluency on Transwell filters was determined fluorometrically using the intracellular pH-sensitive dye 2,7-bis(carboxyethyl)5C6-carboxyfluoresceinacetoxy-methyl ester (BCECF-AM; 5 M; Molecular Rabbit polyclonal to ELMOD2 Probes, Eugene, OR), as described previously (28). Caco-2/BBe/NHE3 cells were exposed to 50 mM NH4Cl during a 45-min dye loading, as described previously (42, 57, 59). Cells were perfused initially with TMA+ solution alone or with 10 M CCh for 1C10 min (130 mM tetramethylammonium chloride, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 25 mM glucose, and 20 mM HEPES, pH 7.4) before being switched to Na+ solution (130 mM NaCl instead of tetramethyl-ammoniumchloride) for the Na+-dependent pHi recovery. At the end of each experiment, the fluorescence ratio was calibrated to pHi using the high potassium/nigericin method. Na+/H+ exchange activity data were calculated as the ratio of Na+-dependent changes in pHi over initial time (pH/min) of Na+-dependent pH recovery using at least three coverslips per condition in a single experiment. Initial rates were analyzed by using Origin (Microcal Software) to determine statistical significance among individual experiments. Means SE were determined from at least three separate experiments. Protein-protein interactions. Protein overlay (Far Western) assays were used to examine the interaction of PLC- purified proteins (2 g of each full-length and -specific array domains) on blots with recombinant c-Src (overlay) by subsequent incubation of blots with monoclonal anti-Src antibody, as described previously (55). For peptide competition studies, PLC- purified proteins were separated on SDS-PAGE, and proteins were transferred to nitrocellulose membranes and exposed to 40 g of either the active or inactive peptides conjugated to BODIPY 577/618 for 1 h at room temperature. Binding of peptides was visualized using a fluorescent Typhoon Imager (Johns Hopkins University School of Medicine Proteomics Core). Membranes were then exposed to 4 g purified full-length Src recombinant protein overnight at 4C. c-Src binding was determined by Western blotting as described above. Results were obtained from three individual experiments. Coimmunoprecipitation. PLC- or NHE3 were immunoprecipitated (IP) from the total lysate of Caco-2/BBe/NHE3 cells (in the presence of 1% Triton X-100). All IPs were done at 4C with constant mixing on a rotary shaker. Briefly, each sample (1 mg of total cell lysate per IP) was first precleared with either protein A-Sepharose beads (Sigma) or protein A beads conjugated to rabbit anti-mouse secondary antibody for 1 h. The precleared lysate was then incubated with 4 g of antibodies to PLC- or c-Src for 1 h. Protein A-Sepharose beads were then added to each IP mixture, and incubation was continued for another 1 h. The beads were washed four times with BIX 01294 phosphate-buffered saline containing 0.1% Tween 20 (Sigma). The IP pellets were analyzed by SDS-PAGE and Western blotted with corresponding antibodies. Caco-2/BBe cells were grown on 10-cm2 Transwell Petri dishes until postconfluent for 12 days and infected with adenovirus 3HA-NHE3 construct as described above. After infection, cells were allowed to recover in normal media for 40 h before CCh treatment. On postconfluency, cells were serum-starved again for 4 h and treated either with vehicle or 10 M CCh (Sigma) for 1C10 min at 37C. Adenovirus infected Caco-2/BBe cells were washed three times in ice-cold phosphate-buffered saline containing 50 mM Tris. Cells were collected and lysed in 500 l of ice-cold lysis buffer (10 mM HEPES, 50 mM NaCl, 5 mM EDTA, 1 mM benzamidine, and 0.5% Triton X-100). Cell lysate was solubilized for 30 min at 4C with end-over-end rotation and subsequently homogenized 10 times using a 23-gauge needle. Cellular debris was cleared by centrifugation at 14,000 rpm for 15 min. Supernatant was incubated with either anti-PLC- or anti-Src antibodies conjugated to protein G agarose beads.The binding curve and statistics were generated using Origin software. PLC-, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC- and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC- SH2 domains, an interaction that was prevented by blocking the PLC- SH2 domain. This study demonstrated that c-Src and subsequently purified with glutathione agarose beads (Sigma). Full-length His6-tagged human recombinant PLC- purified protein was from Calbiochem. Full-length active human c-Src was from Millipore. BODIPY-conjugated peptides. Peptide synthesis and purification were performed by the Synthesis and Sequencing Facility at Johns Hopkins University School of Medicine. Coupling of the peptide to BODIPY 577/618 maleimide was performed according to the manufacturer’s protocol (Invitrogen). PLC- SH2 domain binding (i.e., active) and negative control (i.e., inactive) peptides have been previously described (7, 59). Measurement of Na+/H+ exchange. Cellular Na+/H+ exchange activity in Caco-2/BBe/NHE3 cells grown to 14 days postconfluency on Transwell filters was determined fluorometrically using the intracellular pH-sensitive dye 2,7-bis(carboxyethyl)5C6-carboxyfluoresceinacetoxy-methyl ester (BCECF-AM; 5 M; Molecular Probes, Eugene, OR), as described previously (28). Caco-2/BBe/NHE3 cells were exposed to 50 mM NH4Cl during a 45-min dye loading, as described previously (42, 57, 59). Cells were perfused initially with TMA+ solution alone or with 10 M CCh for 1C10 min (130 mM tetramethylammonium chloride, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 25 mM glucose, and 20 mM HEPES, pH 7.4) before being switched to Na+ solution (130 mM NaCl instead of tetramethyl-ammoniumchloride) for the Na+-dependent pHi recovery. At the end of each experiment, the fluorescence ratio was calibrated to pHi using the high potassium/nigericin method. Na+/H+ exchange activity data were calculated as the ratio of Na+-dependent changes in pHi over initial time (pH/min) of Na+-dependent pH recovery using at least three coverslips per condition in a single experiment. Initial rates were analyzed by using Origin (Microcal Software) to determine statistical significance among individual experiments. Means SE were identified from at least three independent experiments. Protein-protein relationships. Protein overlay (Much Western) assays were used to examine the connection of PLC- purified proteins (2 g of each full-length and -specific array domains) on blots with recombinant c-Src (overlay) by subsequent incubation of blots with monoclonal anti-Src antibody, as explained previously (55). For peptide competition studies, PLC- purified proteins were separated on SDS-PAGE, and proteins were transferred to nitrocellulose membranes and exposed to 40 g of either the active or inactive peptides conjugated to BODIPY 577/618 for 1 h at space temp. Binding of peptides was visualized using a fluorescent Typhoon Imager (Johns Hopkins University or college School of Medicine Proteomics Core). Membranes were then exposed to 4 g purified full-length Src recombinant protein over night at 4C. c-Src binding was determined by Western blotting as explained above. Results were from three individual experiments. Coimmunoprecipitation. PLC- or NHE3 were immunoprecipitated (IP) from the total lysate of Caco-2/BBe/NHE3 cells (in the presence of 1% Triton X-100). All IPs were carried out at 4C with constant mixing on a rotary shaker. Briefly, each sample (1 mg of total cell lysate per IP) was first precleared with either protein A-Sepharose beads (Sigma) or protein A beads conjugated to rabbit anti-mouse secondary antibody for 1 h. The precleared lysate was then incubated with 4 g of antibodies to PLC- or c-Src for 1 h. Protein A-Sepharose beads were then added to each IP combination, and incubation was continued for another 1 h. The beads were washed four instances with phosphate-buffered saline comprising 0.1% Tween 20 (Sigma). The IP pellets were analyzed by SDS-PAGE and Western blotted with related antibodies. Caco-2/BBe cells were cultivated on 10-cm2 Transwell Petri dishes until postconfluent for 12 days and infected with adenovirus 3HA-NHE3 create as explained above. After illness, cells were allowed to recover.Means SE were determined from at least three separate experiments. Protein-protein interactions. NHE3, under basal conditions, an connection that increased rapidly after CCh treatment and occurred before the dissociation of PLC- and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC- SH2 domains, an connection that was prevented by obstructing the PLC- SH2 website. This study shown that c-Src and consequently purified with glutathione agarose beads (Sigma). Full-length His6-tagged human being recombinant PLC- purified protein was from Calbiochem. Full-length active human being c-Src was from Millipore. BODIPY-conjugated peptides. Peptide synthesis and purification were performed from the Synthesis and Sequencing Facility at Johns Hopkins University or BIX 01294 college School of Medicine. Coupling of the peptide to BODIPY 577/618 maleimide was performed according to the manufacturer’s protocol (Invitrogen). PLC- SH2 website binding (i.e., active) and bad control (i.e., inactive) peptides have been previously explained (7, 59). Measurement of Na+/H+ exchange. Cellular Na+/H+ exchange activity in Caco-2/BBe/NHE3 cells cultivated to 14 days postconfluency on Transwell filters was identified fluorometrically using the intracellular pH-sensitive dye 2,7-bis(carboxyethyl)5C6-carboxyfluoresceinacetoxy-methyl ester (BCECF-AM; 5 M; Molecular Probes, Eugene, OR), as explained previously (28). Caco-2/BBe/NHE3 cells were exposed to 50 mM NH4Cl during a 45-min dye loading, as explained previously (42, 57, 59). Cells were perfused in the beginning with TMA+ remedy only or with 10 M CCh for 1C10 min (130 mM tetramethylammonium chloride, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 25 mM glucose, and 20 mM HEPES, pH 7.4) before being switched to Na+ remedy (130 mM NaCl instead of tetramethyl-ammoniumchloride) for the Na+-dependent pHi recovery. At the end of each experiment, the fluorescence percentage was calibrated to pHi using the high potassium/nigericin method. Na+/H+ exchange activity data were determined as the percentage of Na+-dependent changes in pHi over initial time (pH/min) of Na+-dependent pH recovery using at least three coverslips per condition in one experiment. Initial rates were analyzed by using Origin (Microcal Software) to determine statistical significance among individual experiments. Means SE were identified from at least three independent experiments. Protein-protein relationships. Protein overlay (Much Western) assays were used to examine the connection of PLC- purified proteins (2 g of each full-length and -specific array domains) on blots with recombinant c-Src (overlay) by subsequent incubation of blots with monoclonal anti-Src antibody, as explained previously (55). For peptide competition studies, PLC- purified proteins were separated on SDS-PAGE, and proteins were transferred to nitrocellulose membranes and exposed to 40 g of either the active or inactive peptides conjugated to BODIPY 577/618 for 1 h at space temp. Binding of peptides was visualized using a fluorescent Typhoon Imager (Johns Hopkins University or college School of Medicine Proteomics Core). Membranes were then exposed to 4 g purified full-length Src recombinant protein over night at 4C. c-Src binding was determined by Western blotting as explained above. BIX 01294 Results were from three individual experiments. Coimmunoprecipitation. PLC- or NHE3 were immunoprecipitated (IP) from the total lysate of Caco-2/BBe/NHE3 cells (in the presence of 1% Triton X-100). All IPs were carried out at 4C with constant mixing on a rotary shaker. Briefly, each sample (1 mg of total cell lysate per IP) was first precleared with either protein A-Sepharose beads (Sigma) or protein A beads conjugated to rabbit anti-mouse secondary antibody for 1 h. The precleared lysate was then incubated with 4 g of antibodies to PLC- or c-Src for 1 h. Protein A-Sepharose beads were then added to each IP combination, and incubation was continued for another 1 h. The beads were washed four occasions with phosphate-buffered saline made up of 0.1% Tween 20 (Sigma). The IP pellets were analyzed by SDS-PAGE and Western blotted with corresponding antibodies. Caco-2/BBe cells were produced on 10-cm2 Transwell Petri dishes until postconfluent for 12 days and infected with adenovirus 3HA-NHE3 construct as explained above. After contamination, cells were allowed to recover in normal media for 40 h before CCh treatment. On postconfluency, cells were serum-starved again for 4 h and treated either with vehicle or 10 M CCh (Sigma) for 1C10 min at 37C. Adenovirus infected Caco-2/BBe cells were washed three times in ice-cold phosphate-buffered saline made up of 50 mM Tris. Cells were collected and lysed in 500 l of ice-cold lysis buffer (10 mM HEPES, 50 mM NaCl, 5 mM EDTA, 1 mM.
