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. 2021 May 15;22(5):348–365. doi: 10.1631/jzus.B2000441

Fig. 9. Detection of HUVEC cell apoptosis-related protein expression and activity by NC8-pSIP409-alr-ACEIP. (a) Protein profiles of Bcl-2, Bax, and β-actin. β-Actin was used as an internal control, and the induced cells were harvested by centrifugation at 1500 r/min for 3 min at 4 °C and were suspended in 50 mmol/L PBS, followed by sonic disruption. The cell-free extract was analyzed on 12% SDS-PAGE and subjected to western blotting using Bal-2 rabbit polyclonal antibody, Bax rabbit polyclonal antibody, and β-actin mouse monoclonal antibody. (b) The ratio of Bcl-2 to β-actin. (c) The ratio of Bax to β-actin. (d) The ratio of Bcl-2 to Bax. (e‒g) Equal amounts of cell lysates were assayed for caspase-3 (e), -8 (f), and -9 (g) activity using DEVD-pNA, IETD-pNA, and LEHD-pNA as substrates, respectively. The concentrations of fluorescent products released were measured immediately at 405 nm using a microplate reader. Results represent the mean±standard deviation (SD) of triplicate assays. *** P<0.001, compared with the H2O2 positive control group, according to ANOVA. ACEIP: angiotensin-converting enzyme inhibitory peptide; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2-associated X protein; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Ac-DEVD-pNA: acetyl-Asp-Glu-Val-Asp-p-nitroanilide; Ac-IETD-pNA: acetyl-Ile-Glu-Thr-Asp-p-nitroanilide; Ac-LEHD-pNA: acetyl-Leu-Glu-His-Asp-p-nitroanilide; ANOVA: analysis of variance.

Fig. 9