Cytosolic and Nuclear Protein Analysis

Total cytosolic and nuclear extracts were prepared, as previously described [26 (link)], on saphene veins. The following primary antibodies were used: anti- IL-1β (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500 #sc12742, D.B.A, Milan, Italy), anti-TNF-α (Santa Cruz Biotechnology; 1:500 #sc52746), anti-TGFβ1 (Santa Cruz Biotechnology, 1:500 #sc130348, D.B.A, Milan, Italy), anti-VEGF (Santa Cruz Biotechnology; 1:1000 #sc7269), anti- αSMA (Santa Cruz Biotechnology; 1:500 #sc53015) and anti-prolyl endopeptidase (Abcam; 1:1000,#ab58988) in 1× phosphate-buffer saline (Biogenerica srl, Catania, Italy), 5% w/v non-fat dried milk, 0.1% Tween-20 at 4 °C overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA; 1:2000) for 1 h at room temperature. Anti-β-actin (Santa Cruz Biotechnology; 1:1000 #sc47778) and anti-βTubulin (Santa Cruz Biotechnology; 1:1000 #sc5274) antibodies were used as controls. Protein expression was detected by chemiluminescence (ECL) system (Thermo, Waltham, MA, USA), visualized with the ChemiDoc XRS (Bio-Rad, USA), and analyzed by using Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA) as previously reported [27 (link)].

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Casili G., Lanza M., Scuderi S.A., Messina S., Paterniti I., Campolo M, & Esposito E. (2020). The Inhibition of Prolyl Oligopeptidase as New Target to Counteract Chronic Venous Insufficiency: Findings in a Mouse Model. Biomedicines, 8(12), 604.

Publication 2020

anti- IL-1β anti-TNF-α anti-TGFβ1 anti-VEGF anti- αSMA peroxidase-conjugated bovine anti-mouse IgG secondary antibody Anti-β-actin anti-βTubulin ECL) system ChemiDoc XRS Image Lab 3.0 software

Actin Anti igg Antibodies Antibody Bovine Buffer Chemiluminescence Cytosolic Il 1β Membranes Milk Mouse Peroxidase Phosphate Prolyl endopeptidase Protein Saline Tgfβ1 Tnf α Tween 20 Vegf Veins αsma

Corresponding Organization : University of Messina

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Variable analysis

independent variables

Total cytosolic and nuclear extracts were prepared from saphene veins

dependent variables

Protein expression of IL-1β, TNF-α, TGFβ1, VEGF, αSMA, and prolyl endopeptidase

control variables

Protein expression of β-actin and βTubulin

controls

Positive control: Anti-β-actin and anti-βTubulin antibodies were used as controls to ensure the validity of the protein expression results.
Negative control: No information about negative controls is provided.

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