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7 protocols using β actin

1

LPS-Induced Inflammatory Pathway Analysis

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Lipopolysaccharide (LPS, Escherichia coli 0111:B4) was purchased from Sigma (St. Louis, USA). ELISA rat IL-6 and TNF-α kits were purchased from Hefei Bomei Biotechnology CO., LTD (Hefei China). Primary polyclonal antibodies β-actin, HO-1, COX-2, NF-κB, p-NF-Κb, p-ERK1/2, ERK were purchased from Bio Basic Inc., Canada.
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2

Western Blot Analysis of Wound Tissue

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Wound tissues (0.2 g) were harvested and lysed in 2 mL of ice-cold lysis buffer with 2 mmol/L PMSF, 100 mmol/L Na4P2O7, 50 mmol/L HEPES, 10 mmol/L EDTA, 100 mmol/L sodium fluoride, 10 mmpl/L sodium orthovanadate, and protease inhibitor mixture for 15 min. Lysate was centrifuged at 13,000 g for 20 min at 4°C. Proteins denatured in supernatants were electrophoretically separated on an 8% stacking gel and following 12% sodium dodecyl sulfate-polyacrylamide gel and then transferred to 0.2-μm nitrocellulose membranes. The membranes were blocked in TBS-T (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1% Tween 20, pH 7.5) with 5% nonfat milk for 2 hours at room temperature and incubated with rabbit polyclonal antibodies β-actin (1 : 1000), CYP-5 (1 : 1000) (Bio Basic Inc., Canada), HO-1 (1 : 300), TNF-α (1 : 400), IL-6 (1 : 400), CBS (1 : 400), and CSE (1 : 400) (Wuhan Boster Bioengineering Limited Company, China) in TBS-T with 5% nonfat milk overnight at 4°C. After the membranes were extensively washed, a horseradish peroxidase-conjugated secondary anti-rabbit antibody (1 : 10 000 dilution; Sigma Chemical Co., St. Louis, MO, USA) was added and incubated for 2 hours. After being rinsed three times, the membranes were visualized by DAB (Bio Basic Inc., Canada).
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3

Protein Expression Analysis in Granulation Tissue

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Granulation tissues were homogenized and lysed in ice-cold lysis buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, and 1% Triton X-100) containing protease inhibitors (2 mmol/L sodium orthovanadate, 0.2 mmol/L PMSF, 2 μg/mL leupeptin, and 2 μg/mL aprotinin). After centrifuged at 13 000 g for 15 minutes at 4°C, the supernatant was collected. Equal amounts of denatured proteins in the supernatants were separated by 12% SDS-PAGE, and transferred to nitrocellulose membranes. Then the membranes were blocked in TBS-T (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1% Tween20, pH 7.5) containing 5% skim milk for 2 hours. The membranes were incubated with rabbit polyclonal antibodies β-actin, COX-2, VEGF, FGF-2, ERK1/2, p-ERK1/2, Akt, and p-Akt (1: 500) (Bio Basic Inc., Canada) in TBS-T with 5% nonfat milk overnight at 4°C. After the membranes were incubated with anti-rabbit HRP-conjugated antibody (1: 10 000) in TBS-T, target proteins were determined via visualization with DAB (Bio Basic Inc., Canada).
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4

Molecular Markers in Oxidative Stress

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Chloral hydrate, streptozotocin, hemin, and a horseradish peroxidase-conjugated secondary anti-rabbit antibody were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA). Rabbit polyclonal antibodies β-actin, CYP-5 were purchased from Bio Basic Inc. (Canada), and HO-1, TNF-α, IL-6, CBS, and CSE were obtained from Wuhan Boster Bioengineering Limited Company (Wuhan, China).
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5

Pentobarbital Sodium and STZ Induced Rat Model

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Pentobarbital sodium, STZ, and a horseradish peroxidase-conjugated secondary antibody were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA). Rabbit polyclonal antibodies β-actin, COX-2, vascular endothelial growth factor (VEGF), FGF-2, CD34, ERK1/2, p-ERK1/2, Akt, and p-Akt were purchased from Bio Basic Inc. (Canada). Lysozyme-antimicrobial peptide fusion protein was purchased from HebeiChuangyue Biotech. Co., Ltd. (egg-white lysozyme and active fragment of Drosophila melanogaster antimicrobial peptide genes were used to construct fusion genes and subclones) (Langfang, China).
Healthy Sprague-Dawley (SD) rats weighing 220 g to 260 g were purchased from Nanjing Qinglongshan animal breeding ground (Nanjing, China) and raised in our standard animal facility with 22±2°C room temperature and 12-hour day/night alternate. All experimental procedures were handled according to Chinese Community guidelines for the use of experimental animals.
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6

Protein Expression Analysis in Rat Hearts

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Hearts stored at − 70 °C were lysed and homogenized inice-cold lysis buffer (50 mmol/L HEPES, 100 mmol/L sodium pyrophosphate, 10 mmol/L sodium orthovanadate, 100 mmol/L sodium fluoride, 10 mmol/L EDTA, and 1% Triton X-100) containing 2 mmol/L phenylmethyl sulfonyl fluoride. Homogenates were centrifuged at 12,000 g for 20 min at 4 °C. Equal amounts of denatured protein in supernatants were separated by electrophoresis on 12% SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were incubated with rabbit anti-rat primary antibodies such as β-actin, HO-1, COX-2, NF-κB, p-NF-κB, p-ERK1/2, ERK, (Bio Basic Inc., Canada) overnight at 4 °C. Subsequently, membranes were incubated with a horseradish peroxidase-conjugated secondary goat anti-rabbit antibody. After rinsing, proteins were visualized by DAB staining (Bio Basic Inc., Canada).
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7

Lycopene Modulates Oxidative Stress

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Lycopene (502-65-8, 98% purity) was purchased from Nanjing Xinkailong Bioengineering Co., Ltd (Nanjing, China). Carbon tetrachloride (CCl4) was purchased from Suzhou Baiyu Chemical Co., Ltd (Suzhou, China). Antibodies β-actin, heme oxygenase 1 (HO-1), IκB α, p-IκB α, NF-κB, and p-NF-κB were obtained from Bio Basic Inc. (Markham, ON, Canada). Antibodies SIRT 1, peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), microtubule-associated protein 1 light chain 3B (LC3), P62, Beclin 1, regulated in development and DNA damage responses 1 (REDD1), Src homology 2 domain-containing phosphatase 2 (SHP2), transforming growth factor beta (TGF-β), and α-SMA were purchased from Abcam (Cambridge, MA, USA).
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