Role of carrier coating density on PKC signaling induced by binding of anti-ICAM carriers to endothelial cells. (A) Fluorescence microscopy (top panel, with magnified insets showing 1 μm anti-ICAM carriers) and image quantification (bottom panel) of the enrichment of either total PKC or pPKCα upon incubation of anti-ICAM carriers with activated HUVECs at 37°C (enrichment at 10 min and 30 was averaged). Different carrier sizes (250 nm vs. 1 μm) and antibody (Ab) coating densities (7,700 to 13,000 Abs/μm2) are shown. Scale bar = 10 μm (full) or 1 μm (inset). (B) Western blot (top panel) and densitometric quantification (bottom panel) showing pPKCα normalized to GAPDH levels, in cells incubated with anti-ICAM carriers (250 nm or 1 μm in diameter; 9,800 to 30,000 Abs/μm2) as in (A). Mean ± SEM. *Compares carriers of same size but different valency; #compares carriers of same valency but different sizes; (p<0.05 by Student’s t-test).