Figure 7

ICAM-2 WT and variants co-precipitated with α-actinin. A) IP/IB experiments with control cell lines demonstrate the expected association of ICAM-2, α-actinin and actin in lysates from SK-N-ASpIRES.ICAM-2 cells (labeled as WT), but not SK-N-ASpIRESneo cells (neo). Results for these control cell lines were published previously [13]. B) ICAM-2 glycosylation site variants associated simultaneously with α-actinin and actin. Immunoprecipitations (IP) were performed using whole cell lysates and a mouse monoclonal antibody to α-actinin (MAB1682, Millipore). Following protein separation by electrophoresis, immunoblots (IB) were performed with antibodies to α-actinin (sc-7454, Santa Cruz Biotech), ICAM-2 (AF244, R&D Systems), or actin (4968, Cell Signaling). The presence of ICAM-2 WT and variants in each preparation was confirmed by immunoblot analysis of input preparations (a representative blot is shown in Figure 2C) and also by immunoblot analysis of the proteins remaining in the supernatant following precipitation (not shown), to confirm the presence of sufficient/excess ICAM-2 protein or variant in each preparation used for immunoprecipitation experiments.