Anti phospho p44 p42 mapk thr202 tyr204 antibody

C. albicans strains were propagated in YPD medium or supplemented with uridine (80 μg/ml) in an orbital shaker at 30°C. All C. albicans strains generated and used in this study are listed in table S4. SulfoBiotics SSP4 and the H₂S donor GYY4137 were purchased from Dojindo Company. The H₂S probe WSP5 was donated by M. Xian of Washington State University. (−)-Gallocatechin gallate was purchased from MedChemExpress. The fluorescent probes and antifungal drugs were purchased from Sigma-Aldrich. Other ordinary chemical reagents were purchased from Solarbio, Shanghai, China. Anti–phospho-p44/p42 MAPK (Thr²⁰²/Tyr²⁰⁴) antibody was purchased from Cell Signaling Technology (catalog no. 4370S). The antibody against β-tubulin was purchased from Abmart (Shanghai Co. Ltd., catalog no. M30109). The antibody against β-(1,3)-glucan was purchased from Biosupplies Inc. (Parkville, Australia, catalog no. 400-2). Fluoresceine isothiocyanate (FITC)–labeled goat anti-mouse antibodies were purchased from ZSBiO (Beijing, China, catalog no. ZF-0312) or from TransGen Biotech (Beijing, China, catalog no. HS211-01). Tetramethylrhodamine-5-(and-6)-isothiocyanate (TRITC)–labeled goat anti-mouse antibody was purchased from ZSBiO (Beijing, China, catalog no. ZF-0313).

C. albicans cells were harvested by centrifugation, washed once with sterile water, and then resuspended in two volumes of lysis buffer [50 mM tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Triton X-100, and 0.1% (w/v) sodium deoxycholate] containing a protease inhibitor cocktail (Roche) and phosphatase inhibitors (50 mM NaF and 100 mM β-glycerophosphate). One cell volume of 0.4-mm glass beads was added before three 60-s rounds of homogenization in a Precellys 24 homogenizer (Bertin Technologies, Montigny le Bretonneux, France). To equalize the amount of protein loaded, the protein concentration was calculated using Bradford reagent (Beyotime, Shanghai, China) with BSA as a standard. The blots were probed with an anti–phospho-p44/p42 MAPK (Thr²⁰²/Tyr²⁰⁴) antibody (Cell Signaling Technology) for Cek1-P and Mkc1-P detection. A β-tubulin antibody (Abmart Shanghai Co. Ltd.) was used as internal control.