Fig. 3.

AMPK activation inhibits apical pole localization of CRT in mouse S3 proximal tubule cells, as determined by immunofluorescence confocal microscopy. A: time course of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR)-mediated AMPK activation, as measured by immunoblotting of phosphorylated (Thr¹⁷²) AMPK-α (pT172) in S3 cell lysates. AICAR treatment (1 mM) for 2–16 h induced a significant upregulation of phosphorylated (Thr¹⁷²) AMPK-α compared with untreated cells at time 0 under conditions of equivalent protein loading (β-actin control). B: AICAR-induced cytoplasmic redistribution of CRT in polarized S3 cells grown on Transwell filters, as shown by x-z reconstructions of confocal microscope image stacks of S3 cell monolayers immunolabeled using anti-CRT antibody (green) alone or anti-CRT antibody and an anti-ZO-1 antibody (red) grown under control conditions (Con) or in the presence of 1 mM AICAR for 2 h. C: Cy3-coupled phalloidin staining of S3 cell monolayers treated with vehicle (Con) or 1 mM AICAR for 2 h. Images are representative of ≥3 different filter sets and treatments. Scale bar, 10 μm.