BMECs were cultured on Transwells alone or cocultured with hPSC-derived pericyte-like cells, primary brain pericytes, or 3T3s as previously described. After 48 hours of coculture, BMECs were washed once with PBS and lysed with RIPA buffer + Halt protease inhibitor cocktail. The BCA assay was used to determine protein concentration. Proteins were resolved on 4 to 12% tris-glycine gels and transferred to nitrocellulose membranes, which were blocked in tris-buffered saline + 0.1% Tween 20 (TBST) + 5% nonfat dry milk for 1 hour, and incubated with primary antibodies (table S1A) overnight at 4°C. Membranes were washed with TBST (5×) and incubated with donkey anti-mouse or donkey anti-rabbit IRDye 800CW secondary antibodies (LI-COR) for 1 hour. Membranes were washed with TBST (5×) and imaged using LI-COR Odyssey.

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