Characterization of individual single mutants in genes identified by SGA. (A) Calcofluor white (CW) sensitivity. Indicated mutant strains were processed as described in Methods. 3 × 105 cells, and 10-fold serial dilutions thereof were spotted on YPD plates with, and without 25 μg/ml of CW. Plates were incubated at 30°C for 2 days. (B and C) Activation of the CWI pathway. Cell extracts were analysed by Western blot using phospho-p44/p42 MAPK antibodies to detect the dually phosphorylated form of Slt2p, the MAP kinase of the CWI pathway (lower panel, PP-Slt2p). Phosphofructokinase 1 (Pfk1p) was used as a loading control (upper panel, Pfk1p antibodies are directed against both Pfk1p subunits). (B) To analyse constitutive Slt2p phosphorylation, wt and indicated mutant strains were grown at 30°C. (C) To analyse induction of Slt2p phosphorylation in response to different stresses, cells were incubated with CW or at elevated temperatures as described in Methods. For unstressed cells, one representative extract is shown. PP-Slt2p and Pfk1p protein levels were quantified using the ScionImage™ software, and the amount of PP-Slt2p was normalized to the amount of the larger Pfk1p subunit. Values of the mutants were referred to the wt value (set to 1), and are shown below the figure.