Fig. 3. BP180 can co-precipitate actinin-1 from pp126 cells. Extracts of pp126 cells were prepared from cells maintained in medium containing low calcium (lanes 1,2) or cells which had been switched into medium containing 2.0 mM calcium for 4 hours (lanes 3,4). A polyclonal antiserum against the BP180 cytoplasmic domain (J17) (lanes 1,3) was added to the cell extracts which were incubated for 2 hours. No antiserum was included in the extracts shown in lanes 2 and 4. Subsequently protein G-agarose beads were added and, after an additional 2 hours, the beads collected. Precipitated proteins were subjected to SDS-PAGE, transferred to nitrocellulose membranes and processed for immunoblotting using an actinin-1 monoclonal antibody (top). The same immunoblot was then probed with the BP180 antiserum to ensure that BP180 protein had been successfully precipitated (bottom). Note that actinin-1 at 100 kDa is precipitated with BP180 in lane 3 only. An unknown protein recognized by the actinin-1 probe is indicated by an asterisk in lanes 1 and 3 in the top panels. Molecular mass markers indicated on the left of the upper panels are as follows: from top to bottom: 172, 111, 79, 61 kDa.
