Arias et al. BMC Genomics 2011, 12:390 http://www.biomedcentral.com/1471-2164/12/390 Antibiotic susceptibility assays were performed in 96-well sterile plates, filling each well with 95 μl of YPD containing decreasing (twofold) concentrations of the antibiotic nourseothricin, ranging from 500 μg/ml to 0.78 μg/ml (including a growth control without nourseothricin for each serial dilution). Finally, each well (except for sterility controls) was inoculated with 5 μl of a cell suspension containing approximately 104 cells from an exponentially growing culture corre- sponding to each transformed strain under evaluation (overnight growth in SC-Ura and refreshed in YEPD). The plates were incubated in a static culture at 28°C for 40 h, and growth was determined by measuring absorbance at 550 nm in each well with a microplate reader. Each experiment was performed with at least two independent transformants. Western blot assays Yeast cells were grown in YEPD overnight at 24°C to an optical density of 0.8-1 (OD600). The culture was then refreshed to 0.2 OD600 and grown at 24°C for 6 h. The procedures used for immunoblot analyses, including cell collection and lysis, collection of proteins, fractionation by SDS-polyacrylamide gel electrophoresis, and transfer to nitrocellulose membranes, have been described pre- viously [39]. Phosphorylated Slt2/Mpk1 was detected using anti-phospho-p44/p42 MAPK (thr202 /tyr204; Cell Signaling Technology, Beverly, MA). To monitor protein loading, actin levels were determined using mouse anti- actin mAb C4 (ICN Biomedicals, Aurora, OH). For the quantification of the bands from autoradiography films, densitometric analysis was performed using the Quantity One package (Bio-Rad Laboratories). For each sample (mutant strain), a fold-change of Phospho-Slt2 regarding the levels observed in the wild-type strain was calculated as the ratio of the intensity of the P-Slt2 band normal- ized by the amount of actin for each sample (mutant/ wt). Experiments were carried out at least in duplicate. b-galactosidase reporter assays Yeast transformants were grown overnight in an SC-Ura or SC-Ura-Leu medium, as required, at 24°C and then the culture was refreshed in YEPD to 0.2 OD600 and grown at 24°C for 6 h. Yeast cell extracts were prepared by harvest- ing cells by centrifugation from 5 ml of culture. The cells were then resuspended in 250 μl of breaking buffer (100 mM Tris-HCl pH = 8, 1 mM Dithiothreitol, 20% glycerol), and glass beads (Glasperlen ca. 1 mm, Sartorius AG, Ger- many) were added to break cells in a Fast-Prep machine. Finally, extracts were clarified by centrifugation and pro- tein concentrations were measured using the Bradford method. b-galactosidase assays were performed using the crude extracts obtained as described previously [85], scal- ing the protocol to a 96-well microtiter plate format. 5-10 Page 14 of 17 μl of cell extract was mixed with 90-95 μl of Z buffer plus b-mercaptoethanol (0.03%) and 20 μl of o-nitrophenyl-b- D-galactopyranoside (ONPG) (4 mg/ml in Z buffer). The absorbance of the enzymatic reaction was measured at 415 nm on a microplate reader (Model 680, Bio-Rad) after at least 10 min of incubation at 30°C and the addition of 50 μl of 1M Na2CO3 to stop the reaction. b-galactosidase activity was expressed as nmoles of ONPG converted/min- ute/mg of protein. Experiments were performed at least in triplicate involving three independent yeast transformants. Cell wall phenotypic analyses Yeast cells were grown overnight at 24°C in YEPD to mid-log phase. The culture was diluted to an OD600 of around 0.2 and then incubated at 24°C in YEPD for 4 h. These cultures were subsequently diluted again to 0.2 (approximately 15 × 103 cells in 5 μl) and four fivefold dilution series were prepared. Finally, 5 μl of each dilu- tion was spotted on to YEPD solid media containing 100 μg/ml Congo red (Merck), 12 mM caffeine (Sigma), 200 μg/ml SDS (Duchefa Biochemie), 50 μg/ml Hygro- mycin B (Roche), and 40 ng/ml caspofungin (kindly pro- vided by Merck), using a multi-blot replicator (V&P Scientific, San Diego, CA). Growth was monitored on the plates after 2-3 days at 30°C. Flow cytometry and Microscopic analysis Yeast strains were grown overnight at 24°C in YEPD to mid-log phase. The culture was diluted to an OD600 of around 0.2 and then incubated at 24°C in YEPD for 4 h. After this time, 1 ml of cells was collected, washed with PBS and stained with Calcofluor white (Fluorescent Brightener 28, Sigma-Aldrich, St. Louis, MO) at a final concentration of 5 μg/ml for 10 min in darkness. For the flow cytometry analysis of chitin content using Cal- cofluor staining, cells were analyzed with a BD LSR flow cytometer (Becton Dickinson) by acquiring fluorescence through a 380 LP filter. Cell viability was monitored by staining cells with propidium iodide (0.05 mg/ml) acquiring fluorescence through a 670 LP filter. As experimental control, stained yeasts were also analyzed by fluorescence microscopy using a Nikon TE2000 fluorescence inverted microscope equipped with a CCD camera. Digital images were acquired with an Orca C4742-95-12ER camera (Hamamatsu Photonics, Japan) and processed with Aquacosmos Imaging System software. Additional material Additional file 1: Complete set of yeast mutants with increased levels of Slt2 phosphorylation. Representative Western blot experiments of yeast mutants in which MAPK Slt2 was constitutively activated are shown.