Figure 4.

Coimmunoprecipitation of Thy1 and HCN4 by beads coated with anti-Thy1 antibody. Protein G-coated beads (Dynabeads, 100.03D; Invitrogen) were BS³-cross-linked to Thy-1 antibody and then incubated with lysate. Proteins were eluted from these beads, analyzed with Western blot, and sequentially probed for Thy1 (lanes a–d), HCN4 (lanes e–h) using polyclonal antibody, and ABCR (lanes i–l). Before the second and third probes, the previously applied primary antibodies were stripped with glycine. The material aliquoted into each SDS-PAGE gel lane is specified underneath by the combination of + and −. Lanes a, e, and i are material eluted from raw beads (without antibody coating and without lysate). Lanes b, f, and j are material eluted from antibody-coated beads without lysate. Lanes c, g, and k are proteins eluted from Thy1 antibody-coated beads incubated with lysate. Lanes d, h, and l are lysate alone. The migration distances of the molecular weight standards along the left apply to the lanes probed by Thy1 antibody. Those along the right apply to the lanes probed by HCN4 and ABCR antibodies.