Fig. 1. (A) Western blot analysis of Bir1p-GFP expression. Bir1p-GFP cells transformed with either plasmid-borne N-terminally protein-A-tagged Nma111p or Nma111-S235C were grown in glucose medium and subsequently shifted to galactose medium for 24 hours. Bir1p-GFP cells grown in YPAD were used as control cells. Cell lysates were prepared, immunoblotted and probed with a monoclonal anti-GFP antibody. (B) In vitro binding-study of Nma111p and Bir1p. Recombinantly expressed GST-Bir1p and GST were incubated with in vitro-synthesized ³⁵S-labelled Nma111p-fragments. Unbound and bound fractions were analysed by SDS-PAGE and autoradiography. (C) In vitro binding study of Yca1p, Nma111p and Bir1p. Recombinantly expressed GST-Bir1p, GST-Nma111p and GST were incubated with in vitro-synthesized ³⁵S-labelled Yca1p. Unbound and bound fractions were analysed by SDS-PAGE and autoradiography.