Figure 5

Removal of surface glycosaminoglycans (GAGs) blocks PF4 uptake and NF-κB signaling. (A) Serum-starved cells were incubated with various concentrations of the heparan sulfate antagonist Surfen at the indicated concentrations prior to treatment with recombinant PF4 (2 μg/mL) for 30 min. Whole cell lysates were resolved by SDS-PAGE and immunoblots were probed with an anti-PF4 antibody. β-actin is shown as a loading control. Top: bar graph depicts the intracellular PF4 uptake, expressed as the ratio of PF4:β-actin. Data are expressed as mean ± SD and represent three independent experiments. ***, p<0.001, based on ANOVA and Tukey's post-hoc multiple comparison tests. (B) Serum-starved cells were incubated with various concentrations of the heparan sulfate antagonist Surfen at the indicated concentrations prior to treatment with recombinant PF4 (2 μg/mL) for 30 min. Whole cell lysates were resolved by SDS-PAGE and immunoblots were probed with an antibody against phospho-NF-κB p65 (serine 536). Total NF-κB p65 is shown as a loading control. Top: bar graph depicts NF-κB phosphorylation, expressed as the ratio of phospho-NF-κB:NF-κB. Data are expressed as mean ± SD and represent three independent experiments. *, p<0.05, based on ANOVA and Tukey's post-hoc multiple comparison tests. (C) Cells were cultured in the absence (−) or presence (+) of chondroitinase ABC (Chon ABC, 0.5 U/mL) prior to treatment with PF4 (2 μg/mL) for 30 min. Whole cell lysates were resolved by SDS-PAGE and immunoblots were probed with an anti-PF4 antibody. β-actin is shown as a loading control. (D) Confocal micrographs illustrate surface chondroitin sulfate in cells cultured in the absence (−) or presence (+) of chondroitinase ABC, prior to fixation and immunostaining for chondroitin sulfate proteoglycan (CSPG, green) and actin (red). Bar = 20 μm. (E) Cells were cultured in the absence or presence of heparinase III at the indicated concentrations, prior to treatment with PF4 (2 μg/mL) for 30 min. Whole cell lysates were resolved by SDS-PAGE and immunoblots were probed with an anti-PF4 antibody. β-actin is shown as a loading control.