FIGURE 3.

Npr1 protein expression levels were reduced, whereas Npr1 phosphorylation levels were increased in hal4 hal5 mutant strains. A, the indicated BY4741 strains were transformed with a plasmid expressing the Npr1-HA fusion protein, grown to mid-log phase in SD medium supplemented with 0.1 m KCl, and incubated for 30 min in medium supplemented (+) or not (−) with 0.1 m KCl. The amount of Npr1 was analyzed using the anti-HA antibody (top panel), and the amount of protein present in each sample is shown in the Direct blue-stained filter (bottom panel). B, Npr1-HA quantification of the experiment described in panel A. Npr1 protein levels of WT cells grown in potassium supplemented media (+KCl) were used as the reference value. The bars represent the average value for the independent experiments, the circles represent each individual data point, and the error bars show the S.D. C, total RNA was extracted from the indicated strains, and conditions are described in panel A. Northern analysis was performed using a specific probe corresponding to nucleotides 1 to 658 of NPR1 (top panel). The membrane was re-probed with ACT1 as a loading control (bottom panel). D, the NPR1 mRNA signal was normalized using ACT1, and the results are expressed as relative induction of NPR1 using the value corresponding to the WT+KCl sample as the reference value. The bars represent the average value for the independent experiments, the circles represent each individual data point, and the error bars show the S.D. E, the indicated strains from panel A were grown in Translucent medium supplemented with 50 mm KCl. Cells were treated with rapamycin (RAP) (200 ng/ml), cycloheximide (CHX) (25 μg/ml), or incubated for 30 min without potassium and further analyzed for Npr1 electrophoretic mobility. F, Npr1 phosphoshift quantification was performed by densitometry. The position of the Npr1 phosphorylation bands was defined using WT cells treated with cycloheximide. Npr1_P = phosphorylated Npr1. The bars represent the average value for the independent experiments, the circles represent each individual data point, and the error bars show the S.D. Double asterisks (**) indicate statistical significance with a p value < 0.01. Similar results were observed in the W303-1A background.