The lysate was centrifuged at 12 000 rpm for 10 min. The protein extracts were quantified using the Comassie protein assay reagent (Bio-Rad). One hundred and fifty μg of protein was separated on a 10% SDS-PAGE linear gel and then blotted to the nitrocellulose membrane. Before blocking, the MAPK inhibitor equal loading was verified by MemCode ™ Reversible Protein Stain Kit (Pierce) together with the intensity of nonspecific bands. The membrane was then blocked in TBS plus 0.1% Tween 20 and 5 mg/ml dry milk (Carnation) at r.t. for
2 h. The anti-phospho-p44/42 MAPK (selleck chemical Thr202/Tyr204) antibody (New England Biolabs Inc., Hertfordshire, UK) was used to detect phosphorylated forms of Mkc1p and Cek1p MAPKs. The anti-MAPK antibody was used to reveal the total amount of Mkc1p. The anti-Kss1p polyclonal antibody (Santa Cruz Biotechnology), raised in rabbit against Kss1p of S. cerevisiae, was used to detect the total amount of Cek1p. The Act1p signal, obtained using the anti-Act1p antibody (SIGMA), was used as the loading control. Flow cytometry To detect antigen expression, a suspension of 106-107 yeast cells was fixed with 2% paraformaldehyde at
r.t. for 30 min. After washing with ice-cold PBS, samples were incubated at 4°C for 30 min with mAb 1E12 diluted 1:100 and then with a goat SBE-��-CD datasheet anti-mouse IgM-fluorescein-conjugated antibody (Sigma) diluted 1:25. After washing, cells
were immediately analyzed. Fluorescence was analyzed with FACScan flow cytometer (Becton Dickinson, Mountain View, CA) equipped with a 15 mW, 488 nm, air-cooled argon ion laser. FITC fluorescence was measured through a 530 nm band-pass filter and acquired in log mode. Negative controls were obtained by incubating samples with mouse IgM lambda (Sigma). The β-glucan content was expressed in arbitrary units (A.U.) and was calculated as the ratio of the labeled samples on the mean fluorescence channel (mfc) of the corresponding negative controls. The mfc was calculated by Cell Quest software (Becton Dickinson, Mountain View, CA). Cell Vitamin B12 wall components The determination of the sugar monomers, after cell wall polysaccharides extraction with acid hydrolysis, was performed using HPLC with a Dionex Bio-LC system as previously described . Statistics Differences in mean values of analytical determinations were assessed by the Student’s t test, and significance was set at P < 0.05. Results Cell wall integrity To determine the effects of deleting the MP65 gene on the integrity of the cell wall, we tested the mp65Δ mutant for sensitivity to different agents whose effects have been associated with an altered cell wall. The sensitivity was measured by microdilution sensitivity and with solid medium spotting assays.