Fig. 3.

Endogenous pheromone is required for induction by plasma. (A) pCF10 transfer and Asc10 expression of donor strains JRC102(pCF10) or CK104(pCF10). pCF10 transfer and Asc10 expression are induced by plasma in strain CK104 (wild-type) but not in the cCF10-negative strain JRC102. Strain OG1Sp was used as a recipient for mating. For quantification of plasmid transfer, overnight cultures of recipients and donors were diluted 1:10 in M9 medium (with or without 1 ng/ml cCF10) or in plasma and grown for 2 hr before donors and recipients were combined to begin mating. Mating mixtures were incubated for 10 min before plating. Plasmid transfer is depicted as transconjugants per donor. Experiments were done in duplicate; error bars represent 1 SD about the mean. Immunoblot analysis for Asc10 by using a polyclonal anti-Asc10 antibody is depicted below the mating results; samples are in the same order. As for the mating assay, overnight cultures were diluted 1:10 in M9 medium (with or without 1 ng/ml cCF10) or in plasma. Cell extracts were washed twice before harvesting. An equivalent amount of protein was spotted for each sample. (B) Retention of albumin by fractionation of plasma on an iCF10 affinity column. The iCF10 peptide was coupled with the carboxyl terminus to Sepharose 6B. The matrix material was incubated with human plasma and eluted with acetonitrile (see Supporting Text, which is published as supporting information on the PNAS web site). Eluate was separated on an SDS/7.5% PAGE gel and silver stained. Lane 1, human plasma 1:1000; lane 2, acetonitrile eluate from the affinity column; lane 3, purified (commercially obtained) human serum albumin (100 ng).