Yps1-DL has increased shedding activity toward itself and Gas1 compared to Yps1. A) Yeast yps1Δ cells transformed with the indicated plasmids were grown 24 h at either pH 3.0 or pH 6.0. Equal amounts of the concentrated supernatants, without prior treatment with Endo Hf, were applied to 10% SDS-PAGE and immunoblotted with a Gas1 antiserum. B) Cell extracts (C) and concentrated supernatants (S), treated with Endo Hf, were prepared from 24 h cultures at either pH 3.0 or pH 6.0 of yps1Δ yeast transformed with the indicated plasmids. After 10% SDS-PAGE and transfer to nitrocellulose membranes, proteins were immunodetected with an Yps1 antiserum (268–6). Note that 10-times more material was loaded for the extracellular material compared to cell extracts (Representative western from two independent experiments). The percentage of total Yps1-immunoreactive material (%T) found in cell extracts and supernatants is shown below the western blots.