FIG 4.

The balanced regulation of the HOG pathway is critical for environmental stress response and adaptation in C. neoformans. (A and C) Each strain was grown for 16 h at 30°C in liquid YPD medium, 10-fold serially diluted, and spotted onto YPD (glucose-rich) or YP (glucose-starved) medium containing the indicated concentration of KCl or NaCl. (B) WT (strain H99) and ptp1, ptp2, and ptp1 ptp2 mutant strains were grown to mid-logarithmic phase and exposed to 1 M NaCl in YPD medium for the indicated time, and then total proteins were extracted for immunoblot analysis. The Hog1 phosphorylation levels were monitored using anti-dually phosphorylated p38 antibody (P-Hog1; number 4511; Cell Signaling). The same blots were stripped and then reprobed with polyclonal anti-Hog1 antibody for the loading control (Hog1). (C) Each strain was cultured for 16 h at 30°C in liquid YPD medium, 10-fold serially diluted, and spotted onto YPD containing genotoxic stress inducers (110 mM hydroxyurea [HU], 0.04% methyl methanesulfonate [MMS]) or oxidative stress agents (3.5 mM H₂O₂, 0.65 mM tert-butyl hydroperoxide [tBOOH], 2 mM diamide).