FIGURE 6.

Effect of CD44-ICD on the interaction of chondrocyte full-length CD44 with ankyrin adaptor proteins. High density monolayers of RCS or CD44-ICD stable transfectants of RCS (RCS-ICD) chondrocytes were detergent-extracted, and lysates were analyzed by Western blotting directly (lanes labeled Ly) or immunoprecipitated using the anti-CD44-cytotail antibody. Antibody-antigen complexes were captured on protein-G magnetic microbeads, and non-bound flow-through fractions were collected (lanes labeled F) followed by the elution of bound proteins (lanes labeled B). Equivalent volume aliquots of all fractions were processed for Western blot analysis with immunoblotting (IB) performed using HRP-conjugated anti-ankyrin-1 (ANK1; panel A) or anti-ankyrin-3 antibodies (ANK3; panel C, E, and F). After detection, blots were re-probed using HRP-conjugated anti-CD44-cytotail antibody (panels B and D). As a control, the protein-G microbeads were incubated with the anti-CD44-cytotail antibody but without the addition of a cell lysate. The eluted fraction from this control condition (labeled Ctr in panels C and D) was then immunoblotted using HRP-conjugated anti-ANK3 or anti-CD44-cytotail antibodies.