Western Blot for degradation, depletion and protein expression. Mouse brain endothelial cells (bEnd.3) were seeded at 2x10 5 cells/well in 24 well plates and grown for 16-20h prior to treatment. Cells were treated for 24h at 37C with 5% CO2 then washed 3x5min with PBS. Cell lysates were prepared using 1X RIPA (Millipore-Sigma, 20-188) + cOmplete protease inhibitor (Roche, 11836153001) for 5 minutes on ice. Lysates were cleared by centrifugation at 15,000xg for 15 min at 4C. Samples were diluted with 2x Laemelli Sample Buffer (Bio-Rad, 1610737) containing -mercaptoethanol and boiled for 10min prior to loading on AnyKd gel (Bio-Rad, 4568125). SDS-PAGE gels were run and transferred to 0.45m PVDF membrane as previously described (Caianiello, et al). Briefly, gels were run at 120V and transferred in transfer buffer (20% MeOH, 25mM tris base, 192 mM glycine) at 4C for 1h using 300 mA. Blots were blocked for 1h with PBS + 5% BSA. Blots were washed 3x5min with PBS + 0.2% Tween 20 (AmericanBio, AB02038-00500) after each antibody incubation. Gels were imaged using a ChemiDoc imaging system (Bio-Rad). Band intensity was measured using Image J software.