Biotin azide

A photoclickable cholesterol probe (hex-5′-ynyl 3β-hydroxy-6-diazirinyl-5α-cholan-24-oate; Avanti Polar Lipids, AL, USA) was used as described²³ . Briefly, a cholesterol probe stock solution was prepared (2 mmol/L in 50 mg/ml methyl-β-cyclodextrin) and rotated overnight. Endothelial cells and pericytes were co-cultured for 24 hours and treated with lovastatin (5 μg/ml, Merck, Darmstadt, Germany) and t-AUCB (10 μmol/L) for an additional 2 hours. Then cells were incubated with the cholesterol probe (10 μmol/L) for 30 minutes at 37°C. After removing the unbound probe, cells were treated with solvent (0.03% DMSO), 19,20-EDP (3 μmol/L) or 19,20-DHDP (3 μmol/L) in the presence of lovastatin and t-AUCB (10 μmol/L) for 4 hours. After exposure to UV light (λ365 nm) in a crosslinker (Vilber, Eberhardzell, Germany) for 5 minutes, cells were harvested and processed for immunoprecipitation as described above. After immunoprecipitation, the cholesterol probe crosslinked to the proteins of interest was labelled with biotin-azide (Sigma) using a CuAAC-based click chemistry reaction buffer kit according to the manufacturer’s instructions (Jena Bioscience, Jena, Germany).

Fifty μL of NCPs treated with AlkMGO (2b) was added to a premixed solution containing 3 μL of 10 mM biotin azide (Sigma-Aldrich, 762024), 10 μL of a 3:7 mixture of 50 mM CuSO₄, and 100 mM THPTA and vortexed. Thereafter, 5 μL of 100 mM freshly made TCEP was added to initiate the click reaction followed by incubation (1 h) at room temperature. Then, 10 μL of 0.5 M EDTA was added to quench the reactions. Excess reagents were removed by MeOH/CHCl₃ protein precipitation or concentration–dilution using a 0.5 mL Centrifugal Filter (3K, Millipore). The modified histone proteins were enriched by BSA-blocked Streptavidin Magnetic Beads (Thermo Scientific). Next, beads were washed three times with 1× PBS buffer (pH 7.4), boiled, separated on SDS-PAGE, and analyzed by western blot with anti-H3 (Abcam, ab10799) and anti-H4 (CST, 2592S) antibodies.