Figure 4. CD317 interacts with RICH2.... | Rockefeller University Press

Figure 4.

$Figure 4. CD317 interacts with RICH2. (A) Immunoblot probed with an anti-RICH2 polyclonal antibody showing the results of a pull-down assay between a GST-RICH2 fusion protein and a synthetic peptide corresponding to the entire cytosolic N terminus of CD317. Lysate = 10% of total E. coli lysate used in the pull-down; Biotin eluate = GST-RICH2 eluted from biotin-coated beads; CD317 eluate = GST-RICH2 eluted from CD317 peptide-coated beads. (B, top) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (using the indicated antibodies) from lysate of COS cells expressing CD317-GFP and RICH2-RFP (R26.4C antibody detects ZO-1 and was used as a control). (bottom) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (generated using an anti-CD317 antibody) of lysate from polarized control Caco-2 cells or polarized CD317 knockdown Caco-2 cells as indicated. (C) Surface plasmon resonance data from an experiment in which the indicated concentrations of GST-RICH2 were flowed over a streptavidin-coated surface (flow channel) to which biotinylated CD317 N-terminal peptide had been attached. GST-RICH2 at the indicated concentrations was added at 160 s and stopped being added at 320 s. Data represent the binding of GST-RICH2 minus binding to a biotin-blocked control flow channel linked in series to the test channel. (D) Cartoon of the domain organization of RICH2. The region of RICH2 indicated as binding to CD317 was identified because this was the region detected in the initial bacteriophage display screen. The C terminus of RICH2 is a PDZ domain–binding motif, ESTAL. (E) Immunoblot (using an anti-EBP50 antibody) of material immunoprecipitated from lysates of polarized control Caco-2 cells and polarized CD317 knockdown Caco-2 cells (CD317 siRNA) using an anti-CD317 antibody. The top band present in both lanes corresponds to the heavy chain of the anti-CD317 antibody used in the immunoprecipitation. (F) Immunoblot analysis of membrane and cytosol fractions from Caco-2 cells using antibodies to RICH2 and the integral membrane protein CD99 as indicated. (A, B, E, and F) Molecular mass is indicated in kilodaltons.$

CD317 interacts with RICH2. (A) Immunoblot probed with an anti-RICH2 polyclonal antibody showing the results of a pull-down assay between a GST-RICH2 fusion protein and a synthetic peptide corresponding to the entire cytosolic N terminus of CD317. Lysate = 10% of total E. coli lysate used in the pull-down; Biotin eluate = GST-RICH2 eluted from biotin-coated beads; CD317 eluate = GST-RICH2 eluted from CD317 peptide-coated beads. (B, top) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (using the indicated antibodies) from lysate of COS cells expressing CD317-GFP and RICH2-RFP (R26.4C antibody detects ZO-1 and was used as a control). (bottom) Immunoblot (using an anti-RICH2 antibody) of immunoprecipitates (generated using an anti-CD317 antibody) of lysate from polarized control Caco-2 cells or polarized CD317 knockdown Caco-2 cells as indicated. (C) Surface plasmon resonance data from an experiment in which the indicated concentrations of GST-RICH2 were flowed over a streptavidin-coated surface (flow channel) to which biotinylated CD317 N-terminal peptide had been attached. GST-RICH2 at the indicated concentrations was added at 160 s and stopped being added at 320 s. Data represent the binding of GST-RICH2 minus binding to a biotin-blocked control flow channel linked in series to the test channel. (D) Cartoon of the domain organization of RICH2. The region of RICH2 indicated as binding to CD317 was identified because this was the region detected in the initial bacteriophage display screen. The C terminus of RICH2 is a PDZ domain–binding motif, ESTAL. (E) Immunoblot (using an anti-EBP50 antibody) of material immunoprecipitated from lysates of polarized control Caco-2 cells and polarized CD317 knockdown Caco-2 cells (CD317 siRNA) using an anti-CD317 antibody. The top band present in both lanes corresponds to the heavy chain of the anti-CD317 antibody used in the immunoprecipitation. (F) Immunoblot analysis of membrane and cytosol fractions from Caco-2 cells using antibodies to RICH2 and the integral membrane protein CD99 as indicated. (A, B, E, and F) Molecular mass is indicated in kilodaltons.