Partial pro-peptide removal is essential for binding of Yps1-DL to a Pepstatin A-agarose resin. A) Yeast yps1Δ cells transformed with the indicated plasmids were grown at either pH 3.0 or pH 6.0 for 24 h and equal amounts of concentrated media (7.7 × 107 cells equivalent for Yps1-DL and 7.7 × 106 cells equivalent for ssYps1-DL) were treated with Endo Hf and the proteins were separated by 10% SDS-PAGE. After transfer to PVDF membranes, immunoblotting was performed with an Yps1 antiserum (268–6) (Representative western from three independent experiments). B) Yeast yps1Δ cells transformed with ssYps1-DL were grown 24 h at pH 6.0 and the concentrated supernatant was applied to a Pepstatin A-agarose resin as described in “Methods”. Aliquots of the starting material (Input), the unbound material (Flow-through) and the eluted fractions (Fraction 1 & 2) were then separated by 10% SDS-PAGE and immunoblotted with an Yps1 antiserum (268–6) (Representative western from two independent experiments).