St360

Cortical neurons were pretreated with mithramycin A32 (link) (Sangon Biotech M0668) 300 nM or pifithrin-α³¹ (Sigma P4236) 10 μM 24 h before preconditioning. Neurons were pretreated with NADPH45 (link) (Beyotime ST360) 10 μM or N-acetyl-L-cysteine33 (link) (NAC, Sigma A9165) 100 μM 30 min before preconditioning.

Superoxide levels in thoracic and abdominal aorta rings were assessed by lucigenin-enhanced chemiluminescence as previously documented [25 (link)]. Samples were added to polypropylene cuvettes with PBS and lucigenin (5 μmol/L). After 10 min of dark adaptation, basal chemiluminescence was measured for 5 min using a luminometer (FB15, Zylux, Maryville, TN, USA). For measurement of NOX activity, luminescence was assessed using NADH (0.2 and 1 mmol/L, ST358, Beyotime, Shanghai, China) or NADPH (0.2 and 1 mmol/L, ST360, Beyotime) as substrates. The source of superoxide was determined by treating samples with apocynin (targeting NOX, 100 µmol/L, 178385, Sigma-Aldrich), L-NAME (targeting eNOS, 100 µmol/L) or oxypurinol (targeting XO, 100 µmol/L, O6881, Sigma-Aldrich) for 30 min prior to chemiluminescence measurement. Relative light unit (RLU) values were normalized to the surface area of each vessel.