FIGURE 2:

Pressure effects on WNK3 autophosphorylation and activity in vitro. (A) using pWNK and gOSR. in the absence or presence of added hydrostatic pressure. Pro-Q® stain (back) of phospho-proteins pWNK3 and phopho-gOSR. Triplicated 15-min reactions –/+ 190 kPa using 4 mM uWNK3 and 40 mM gOSR1 in 150 mM Cl^–, run at 25°C. The blue bands are Coomassie staining as loading controls. In the bar graph, gray corresponds to unpressurized pWNK3 and phospho-gOSR; red, under pressure. Two-sided t test was conducted for each protein giving * p < 0.01, *** p < 0.0001. (B) ADP-Glo ® measuring ATP consumption using uWNK3 (4 mM) and gOSR1 (40 mM). Reactions were run at 30°C. Appearance of ADP without (white) and with (blue) 190 kPa measured at 10 min and 15 min (background from reagents subtracted). (C) g³²P ATP spot blots imaged by false color autoradiography of autophosphorylation of 40 mM uWNK3 at 0 and 190 kPa hydrostatic pressure induced with N₂ gas. p = 0.007.