Fig. 2. Immunoprecipitation studies indicate that BP180 can bind actinin-4 and actinin-1 but not MRLC. Approximately 1 µg each of either HA-tagged actinin-4 (Ai), actinin-1 (Aii) or MRLC (Aiii) were incubated with a purified S-tagged BP180 fusion polypeptide in Hepes-buffered saline with 1% Brij 97 and 0.2% SDS at 4°C for 2 hours. As a control, tagged BP180 polypeptide was omitted from the immunoprecipitation mixture (Ai-iii, lane 2). A polyclonal antiserum against the BP180 cytoplasmic domain was added to the protein mixture for an additional 2 hours, followed by the addition of protein G-agarose beads (Life Technologies/BRL). The precipitated proteins were subjected to SDS-PAGE and transferred to nitrocellulose membrane. The blot was probed either with HA.11 antibody directed against the HA tag (A) or an alkaline phosphatase-conjugated S-protein that binds S-tag peptide fusion proteins (B). In lane 1 in Ai, the HA.11 antibody recognizes a single polypeptide, indicating that actinin-4 has been precipitated with BP180. Similar results were obtained when BP180 was precipitated from the BP180/actinin-1 mix (lane 1 in Aii). By contrast, MRLC was not precipitated from the MRLC/BP180 mix as indicated by the absence of a band in lane 1 in Aiii. No protein is recognized by the HA.11 antibody in Ai-iii lane 2, indicating that actinin-4, actinin-1 or MRLC were not precipitated by the BP180 antiserum. The enzyme-linked S-protein in B recognizes the immunoprecipitated BP180 polypeptide in Bi-iii lane 1. Molecular mass markers indicated on the left of the upper panels are as follows: from top to bottom 172, 111, 79, 61, 49, 36 kDa.
