Figure 5.

Coimmunoprecipitation of Thy1 and HCN4 by incubation of beads, coated with monoclonal anti-HCN4 antibody, with lysates, formatted as in Figure 4. The proteins eluted from these beads were separated by SDS-PAGE, with samples of the lysate and molecular weight standards in adjacent lanes. The gels were analyzed with Western blot and the blots were separated into three segments by slicing at the level of the 185 kD standard and at approximately 50 kD. The upper, middle, and lower segments were probed with antibodies against ABCR, HCN4 (polyclonal), and Thy1, respectively. The ABCR and Thy1 antibodies were visualized with Fc fragment-specific, DyLight 649-conjugated, goat anti-mouse IgG. The HCN4 antibody was visualized with detection reagent (Clean-Blot IP; Thermo Scientific).