Figure 8.

Comparison of HA binding by the native and recombinant 175-kDa HARE proteins. Membranes from isolated LECs (lanes 1 and 2) and SK-175HARE-34 cells (lanes 3 and 4) were solubilized in TBS containing 0.5% NP-40 plus protease inhibitors and HARE proteins were immunoprecipitated using mAb-30 coupled to Sepharose. The proteins were eluted with sample buffer (Laemmli, 1970), subjected to SDS-PAGE and electrotransfer, and the nitrocellulose was incubated overnight in TBS containing 0.1% Tween 20. Ligand blotting with 1 μg/ml ¹²⁵I-HA (lanes 1 and 3 from autoradiogram) was performed as described in MATERIALS AND METHODS. The same blots were then incubated in TBS containing 1% BSA and subjected to Western analysis (lanes 2 and 4) by using a mixture of eight mAbs against HARE. A series of dilutions verified that the Western staining responses for both samples were proportional to protein load and were not saturated. The open and solid arrows indicate, respectively, the ∼300- and 175-kDa HARE species. The HA-binding intensity relative to the Western staining of the 175-kDa HARE was essentially the same from LECs and the stable cells.