Apoptosis whole cell lysate kit

Plasma EVs from above were lysed using M-PER™ Reagent (Thermo Scientific), supplemented with protease and phosphatase inhibitors (Roche). Equal volume of EV lysates were used to quantitatively measure proteins in the following Meso Scale kits (Meso Scale Diagnostics): total p53, phospho-p53 (Ser15), PARP (cleaved), and Caspase-3 (cleaved) by the Apoptosis Whole Cell Lysate Kit (Cat#K15102D-1); phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), and phospho-ERK1/2 (Thr/Tyr: 202/204; 185/187) by the Apoptosis Whole Cell Lysate Kit (Cat# K15101D-1) and a custom made kit for phospho-Tau181 kit. Meso Scale kits were analyzed on a Meso Quickplex SQ 120. LC3 was measured using an ELISA kit (Cat# MB-S2602917 MyBioSource, Inc.). Clusterin was quantified by the Clusterin Competitive ELISA Kit (Cat# AG-45A-0013YEK-KI01 Adipogen Corporation).

HRMEC were seeded onto gelatin‐coated 60 mm diameter dishes. On the next day, they were treated for 24 hours with PBS or 100 ng/mL NGF in plain EGM under normoxia (21% O₂) or hypoxia (1% O₂). The samples were washed twice with cold PBS and lysed in RIPA Buffer (Santa Cruz, Heidelberg, Germany) including phosphatase, protein inhibitors (Roche, Mannheim, Germany) and benzonase (Sigma‐Aldrich). Lysates were collected and protein concentration was determined with the BCA Protein Assay kit (Invitrogen). Cleaved PARP levels were studied in equal amounts of protein lysates with the Apoptosis Whole Cell Lysate Kit (Meso Scale Diagnostics, Rockville, MD, USA). Samples were then analysed in a plate reader (QuickPlex SQ 120; Meso Scale Diagnostics) following the manufacturer's protocol.