Bax Antibody (4H32): sc-70407

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:2000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:2000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

The HUVECs were collected and lysed in RIPA lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) containing protease and phosphatase inhibitor cocktail. The protein concentration was determined using a BCA protein content kit (Beyotime Institute of Biotechnology). Proteins (40 µg) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with the following appropriate primary antibodies overnight at 4°C: Bcl-2, Bax; cytochrome c, COX IV, cleaved caspase-9, cleaved caspase-3, cleaved caspase-8, cleaved-PARP and β-actin. Following incubation with the secondary antibodies including horseradish peroxidase-conjugated rabbit anti-mouse (cat. no. 58802; 1:1,000; Cell Signaling Technology, Inc.) or horseradish peroxidase-conjugated donkey anti-goat (cat. no. sc-2056; 1:1,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature, the signals were determined using an ECL kit and the intensity of the protein bands was analyzed using ImageJ software (version 1.41; National Institutes of Health, Bethesda, MA, USA).

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