Figure 2. Steady laminar flow, but not oscillating disturbed flow, decreased EC eNOS O-GlcNAcylation.

(A) Western blot for all O-GlcNAcylated proteins, with quantification of total GlcNAcylated proteins and GlcNAcylation of the ~140 kDa protein only (indicated by arrow). Confluent HUVEC were first adapted to static culture (‘S’), steady laminar (‘L’, 20 dynes/cm2), or oscillating disturbed flow (‘D’, 4±6 dynes/cm²) for 24 hrs. n = 14 – 31 samples, normalized to static culture and GAPDH. For GlcNAc-eNOS, p = 1.84×10⁻¹¹ by one-way ANOVA, with p values on the figure determined by Tukey’s multiple comparisons test. (B) Western blot for all O-GlcNAcylated proteins and total eNOS for HUVEC with eNOS knocked down via siRNA. Quantification of GlcNAcylation of the ~140 kDa protein and total eNOS. (No: HUVEC without siRNA; Neg: control siRNA; E: eNOS siRNA). n = 3 samples. For GlcNac/GAPDH and eNOS/GAPDH, p = 3.57×10⁻³ by Kruskal-Wallis test, with p values on the figure determined by Dunn’s multiple comparisons test (all samples compared to “No”). (C) Western blot for all O-GlcNAcylated proteins and total eNOS for HUVEC after eNOS immunoprecipitation. Static ‘S’, steady laminar ‘L’, 3 hour 5 mM glucosamine ‘G’. 3 independent samples were combined into 1 sample to obtain enough protein for immunoprecipitation.