Fig. 2. MAP kinases MPK4 and MPK12 inhibit HT1-mediated CBC1 kinase phosphorylation in the presence of elevated NaHCO₃ in vitro.

(A) Recombinant HT1 and CBC1 proteins were incubated with MPK4, MPK12, or the (pseudo)-kinase domain of GHR1 in the presence or absence of 20 mM NaHCO₃ for 30 min, and in vitro phosphorylation assays were performed. Histone was used as an artificial protein kinase substrate. (B) MPK4, HT1, and CBC1 proteins were incubated with NaHCO₃ at the indicated concentrations for 30 min, and in vitro phosphorylation assays were performed. (C) CBC1 band intensities as shown in (B) were measured using ImageJ. n = 4 experiments. Error bars show ±SD. (D) MPK4, HT1, and CBC1 proteins were incubated in reaction buffers adjusted at different pH (6.5 to 8.5) for 30 min, and in vitro phosphorylation assays were performed. (E) CBC1 band intensities and the density ratios of “+NaHCO₃” to “−NaHCO₃” (= +NaCl controls) for each pH condition as shown in (D) were measured using ImageJ. n = 4 experiments. Error bars show ±SD.