Extended Data Fig. 4: Luminal cerebrovascular proteomics does not reveal substantial downregulation of mucin-domain glycoprotein scaffolds with age.
From: Glycocalyx dysregulation impairs blood–brain barrier in ageing and disease
a) Luminal cerebrovascular proteomics scheme. b) Western blot of brain microvessel lysates from mice perfused with PBS or sulfo-NHS-biotin detected via streptavidin. In, input; FT, flowthrough; E, eluate. c) Cerebrovascular labeling by sulfo-NHS-biotin detected via streptavidin. Scale bar = 25 µm. d) Gene ontology (GO) term cellular component and biological process analysis of all proteins detected in luminal cerebrovascular proteomics experiment. e) Principal component analysis (PCA) of proteomic samples from young (3-month-old) versus aged (21-month-old) mice (n = 6 mice per group). f) Volcano plot of luminal cerebrovascular proteins identified in young (3-month-old) versus aged (21-month-old) mice (n = 6 mice per group). Peptide and protein identifications were filtered to a 1% FDR. Notable mucin-domain glycoproteins are highlighted in purple. Proteins only detected in young animals (in ≥ 50% of young animals) are highlighted in blue in the upper lefthand corner. Proteins only detected in aged animals (in ≥ 50% of aged animals) are highlighted in red in the upper righthand corner. g) NID1 expression in cortical vessels in young (3-month-old) versus aged (21-month-old) mice. Areas with poorly colocalized NID1 and StcEE447D-AF647 signal are indicated by the white triangles. Scale bar = 50 µm. h) Quantification of NID1 expression in COL4A+ vasculature in (g) (n = 3 mice per group; two-sided t-test; mean ± s.e.m.). i) Quantification of vascular NID1 colocalization with StcEE447D and COL4A via Pearson correlation (n = 8 images from aged mouse group; two-sided paired t-test; mean ± s.e.m.).
