Figure 6. Effect of hyperosmolarity and A769662 treatment on AMPK and SPAK phosphorylation in mouse RBCs.

Washed mouse erythrocytes were incubated at 5% haematocrit and 37°C in Hepes-buffered Krebs medium containing 11 mm glucose, with or without 20 μm A769662 and with or without 0.3 m sucrose for 5 or 60 min. A, upper panel, a representative immunoblot of α-AMPK Thr172 phosphorylation versus total α1-AMPK. B, upper panels, representative immunoblots of SPAK Thr233 and Ser373 phosphorylation versus total AMPK as a loading control. Lower panels, densitometric scanning ratios of band intensities obtained with the anti-phospho antibodies relative to those obtained for the loading controls; the results are the means ±s.e.m. of three independent experiments. *Significant difference (P < 0.05, paired t test) with respect to the controls.