Figure 4. In situ [³²P]-orthophosphate labeling of NHE1 during cell suspension in hyperosmotic medium.

(upper) ³²P incorporation into immunoprecipitated NHE1 from atRBCs. autoradiograph images corresponding to immunoprecipitated NHE1 from membrane fractions of [³²P]-orthophosphate labeled atRBCs suspended in isosmotic medium, or hyperosmotic media (n1.6×IR) with or without CLA treatment. Below each autoradiograph band is the corresponding NHE1 Western blot band detected on the same PVDF membrane. (lower) A quantitative comparison of data from the autoradiograph bands described in panel A (normalized to NHE1 Western blots from the same PVDF membrane). Relative phosphorylation of immunoprecipitated NHE1 is significantly increased by suspension of cells in hyperosmotic (n1.6×IR) solution with or without CLA treatment relative to the isosmotic control (*p<0.05 , **p<0.01; n = 6±SEM). No significant difference is detected between the two treatment conditions (hyperosmotic ± CLA; n.s.).