Figure 3
Acute high shear stress and activation of the NF-κB classical pathway—in vitro. The NF-κB classical pathway was activated under acute high shear stress as indicated firstly by the nuclear translocation of NF-κB and reciprocal reduction in cytosolic levels of IκB⍺, as well as phosphorylation and increased DNA binding affinity. HUVECs were exposed to acute arterial WSS for 30 and 90 min or maintained in static conditions, then cellular fractions were collected to be analysed by Western blotting. Total amounts of protein were quantified relative to stain-free loading controls and expressed as a fold change of static control. (A) Nuclear NF-κB, (B) cytosolic levels of IκB⍺ and (C) Phospho-NF-κB (Serine residue 276 (Ser276)) from whole cell lysates were analysed by WB, and normalised to stain-free loading controls from nuclear, cytosolic and whole cell lysates, respectively. Western blots shown are representative of 5 or 6 independent experiments showing, in addition to NF-κB and IκB⍺, markers of nuclear and cytosolic fractions, Lamin A/C and GAPDH respectively (D). The transcriptional activation of NF-κB under acute arterial WSS, for both 30 and 90 min, was assessed by phosphorylation at Serine residue 276 (C) and DNA binding activity (E) in whole cell lysates. ELISA based DNA binding activity assay was performed using total cell lysates and was expressed relative to static controls and were representative of 3 independent experiments. One-way ANOVA followed by post-hoc pairwise comparisons with Bonferroni correction was used to calculate significance, where, ** indicates p < 0.01 and * indicates p < 0.05.
