FIG 5.
Biochemical assessment of the avidity of CDV H-cSLAM interactions. (A to C) Coimmunoprecipitation assays. CDV HFLAG and HA-tagged wt cSLAM, mutant cSLAM, or lion SLAM were coexpressed in Vero cells and subsequently lysed with RIPA buffer 24 h posttransfection. The CDV H-SLAM complexes were then immunoprecipitated (IP) with an anti-HA MAb and protein G-Sepharose bead treatment. Proteins were boiled and subjected to immunoblotting (IB) using an anti-CDV H polyclonal antibody to detect H antigenic materials (co-IP). Co-IP CDV H proteins were detected in comparison with CDV H proteins present in cell lysates prior to IP by immunoblotting using the same anti-H antibody. Gels illustrating total expression and immunoprecipitation of SLAM molecules are also shown (detected using an anti-HA polyclonal antibody). The specific MAbs used for the immunoprecipitation (IP) or immunoblotting (IB) steps are indicated on the left of the gels. (B) Of note, the white line on the right side of the gels shows where we cropped the gels for easier comparisons between the canine SLAM mutants and the lion SLAM molecule.