FIG 2.

Bem4p is a pathway-specific regulator of the filamentous-growth pathway. (A) (Left) Colony ruffling as an indicator of filamentous growth. Bar, 1 cm. (Middle) Halo assay as a measure of growth arrest in response to α-factor. (Right) Growth of cells in YEPD plus 1 M KCl. Images from the 2-day time point are shown. (B) β-Galactosidase activity of the mating pathway FUS1-lacZ reporter. Cells were grown to mid-log phase in YEPD, washed, and treated with 1 mM α-factor for the indicated times. Values are the averages from two independent trials; error bars show the standard deviations between trials, and asterisks denote P values of <0.05. (C) Co-IP analysis of Bem4p-HA with Kss1p-GFP, Fus3p-GFP, and Hog1p-GFP. Bem4p-HA was overexpressed in cells containing genomic fusions to GFP. Band intensity relative to input levels after background subtraction was determined by ImageJ. Input levels are 10% for WCE. (D) Role of high-copy-number Bem4p-GFP in regulating the mating pathway. Cells were grown to mid-log phase in YEPD and induced by 5 μM pheromone and evaluated by immunoblotting for phosphorylation of Kss1p and Fus3p. (E) Role of high-copy-number Bem4p-GFP in activation of the HOG pathway. Cells were grown to mid-log phase in YEPD and induced by 0.4 M KCl at the indicated times for phosphorylation of Hog1p.