Alexa fluor 680 nm dye

vWF and D-Dimer in the plasma were measured by Western blot at age 5 months. Equal volumes of the plasma were loaded on the gel. Before loading, 3× Laemmli Sample buffer supplemented with dithiothreitol (no. 7722, Cell Signaling) was added to the samples and the mixtures were boiled for 5 minutes. Immunoblots used primary antibodies against vWF (no. A0082, Dako) and D-Dimer (no. bs-3514R, Bioss) and secondary antibodies labeled with Alexa Fluor 680 nm Dye (Invitrogen). The immunoblots were scanned and integral fluorescence from each band was measured using Odyssey Infrared Imaging System (Li-COR Biosciences). See complete unedited blots in the supplemental material. For final quantification, we corrected the measurements for plasma dilution with sodium citrate by multiplying by the coefficient of dilution calculated as described in the Mouse blood collection and plasma isolation section.

Cells were rinsed with phosphate-buffered saline (PBS), adjusted with NaCl to the same osmolality as the medium, then lysed with RIPA buffer (50 mM Tris-HCl, 1% NP-40, 150mM NaCl, 1mM EDTA, 1mM NaF, 1 mM Na₃VO₄, and protease inhibitors (Roche Diagnostics, Indianapolis, IN)). 3X reducing Laemmli Sample buffer (No. 7722, Cell Signaling, Danvers, MA) was added to the lysates and samples were boiled for 5 minutes. Sample loading onto gels was equalized according to the total protein concentration measured before addition of Laemmli buffer. Primary antibodies were against VCAM-1 (No. 305801, BioLegend, San Diego, CA), alfa-tubulin (No. 691251, MP Biomedicals, Solon, OH). Secondary antibodies were labelled with Alexa Fluor 680 nm Dye (Invitrogen, Carlsbad, CA). Immunoblots were scanned and Integral Fluorescence (IF) from each band was measured using Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE).