FIGURE 6.

Effects of changes in extracellular sodium on native SNAT2 protein expression in HeLa cells. (A) HeLa cells were maintained in the absence or presence of a 1× physiological AA mix for 8 h in HEPES buffer (HBS) containing 140 mM NaCl (Na-HBS). Cells were either harvested or incubated for a further 4 or 6 h in AA-deficient Na-HBS, or HBS in which NaCl was replaced with 50 or 100% TMACl. At the end of these periods cells were harvested and total membranes (10 μg) prepared, which were subjected to SDS–PAGE and were then immunoblotted with antibodies against SNAT2 or the beta-subunit of the insulin receptor (IRβ). (B) HeLa cells were incubated for an initial 8 h with or without a 1× AA mix in Na-HBS and then harvested or incubated for an additional 6 h without AAs in a partial (50%) sodium-replaced, or fully (100%) sodium-replaced HBS media. Sodium was partially or fully replaced with either TMA, NMDG, or choline. At the end of the indicated incubation periods indicated, cells were harvested and total membranes (10 μg) prepared before SDS–PAGE and immunoblotting against SNAT2 and IRβ (used as a loading control). Endogenous SNAT2 expression was quantified using ImageJ software and results expressed as relative abundance with respect to the IRβ. Blots are representative of three separate experiments, bar graphs values are mean ± SEM (n = 3). Asterisks indicate a significant difference between the indicated bars (P ≤ 0.05).