Fig. 3. Byr4p is degraded if its interaction with Spg1p is compromised. (A) Protein extracts (EX) were prepared from the indicated strains and passed over a glutathione-agarose column. The flow-through (FT) and three successive eluates (E1, E2, E3) from the column were analysed using antibodies against Byr4p or GST. (B) Protein extracts were prepared from the indicated strains, grown at 25°C and western blots were probed with antibodies against Byr4p, GFP and tubulin. The relevant genotypes of the strains are shown above the panel. When two copies of spg1 are present, the first indicates the gene inserted into leu1, the second the state of the native spg1 locus. (C) Protein extracts from the indicated strain were analysed as described in A. (D) Cells from the indicated strains were shifted from exponential growth in YE medium at 25°C to 36°C for 4 hours before harvesting the cells. Protein extracts were prepared and western blots were probed with the indicated antibodies. The western blot on the right of panel D shows the mts3-1 sample untreated (E), treated with alkaline phosphatase (+P) or with alkaline phosphatase in the presence of phosphatase inhibitors (M). The arrows indicate the positions of the three forms as defined in Fig. 2A.