Fig. 7. Effects of high glucose concentration on the expression of PC. (A) Western blotting of PC expression in HGEC cultured in the presence of 5 mM or 25 mM glucose. Equal amounts of total protein (50 µg) were analyzed and blotted with 3D3 anti-PC antibody. Blots were stripped and reprobed with anti-tubulin antibody to verify equal loading. (B) Northern blot analysis of PC mRNA in HGEC. Total RNA was isolated from cells grown in 5 mM or 25 mM glucose. 10 µg of each sample were probed with [³²P]dCTP-labeled human PC-cDNA. Filters were rehybridized with [³²P]dCTP-labeled cDNA probe for GAPDH to verify equal loading. (C) FACS analysis of PC surface expression in HGEC cultured in 5 mM (unshaded histogram) or 25 mM glucose (shaded histogram). First peaks (interrupted lines) represent cells (cultured in 5 mM or 25 mM glucose) incubated only with FITC-conjugated IgG. Mean of fluorescence and percentage of positive cells was calculated in the histogram section indicated as M1. (D) Western blotting of PC expression in HGEC cultured on dishes either uncoated (lane 2) or coated with 20 µg/cm² of each laminin (lane 3), or GBM (lane 4), in the presence of 25 mM glucose. Control cells were cultured on plastic in the presence of 5 mM glucose (lane 1). (E) PC expression in streptozotocin-treated diabetic rats. Micrographs represent PC staining in control (upper panel) or diabetic (lower panel) glomerulus. Scale bar, 50 µm.