Proteins from HUVECs were isolated with protein extraction buffer (Thermo Fisher) by incubation on ice for 30 min. After centrifugation at 12,000 rpm for 15 min at 4 °C, supernatants were collected. Protein concentrations were determined by a BCA protein assay kit (Thermo Fisher). Western blotting was performed as described previously [20 (link)]. In brief, Total 20 μg of protein samples were loaded at SDS-PAGE electrophoresis gel for running. Then the protein was transferred to NC membrane and blocked in 5% milk powder. Membranes were incubated with specific primary antibodies [anti-BCL2 antibody (Rabbit, 1:1000 dilution), anti-BAX antibody (Rabbit, 1:1000 dilution), anti-HDAC4 antibody (Rabbit, 1:1000 dilution), anti-GAPDH antibody (mouse, 1:1000 dilution) and anti-β-Actin antibody (mouse, 1:1000 dilution), from Cell Signaling Technology] at 4 °C overnight. Then the membranes were washed and incubated with a secondary antibody (Li-COR Biosciences, Lincoln, NE) conjugated with IRD800 at room temperature for 1 h in the dark. The membranes were then washed and analyzed by the Odyssey software system (Li-COR). The ratio of the protein change was normalized to GAPDH.
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