Hvlp04700

Protein lysis (R0010; Solarbio) was added to CD34⁺ cells to isolate total protein, which was then separated using 12% SDS-polyacrylamide gel electrophoresis (P1200; Solarbio) and loaded onto a polyethylene difluoride membrane (HVLP04700; Millipore). Next, nonfat milk-blocked membranes was added with primary antibodies RAC1 (1:1000; 05-389; MilliporeSigma), CREB (1:1000; 9198; Cell Signaling Technology), p-CREB (1:1000; 9197; Cell Signaling Technology), PD-L1 (1:1000; ab205921) Abcam), Bcl-2 (1:1000; ab32124; Abcam), Bax (1:1000; ab32503; Abcam) and GAPDH (1:1000; ab8245; Abcam) overnight at 4°C, and supplemented with horseradish peroxidase-conjugated secondary antibody (1:2000, ab6721; Abcam) for 2 h. Following diaminobenzidine development, imaging was done under Gel Doc Xr (Bio-Rad).

Ultra-pure water (<18 MΩ cm < 5 μgL⁻¹ TOC) was obtained from a purification system Arium 61316-RO plus Arium 611 UV (Sartorius, Germany). Methanol (HPLC grade) and formic acid (puriss. p. a. for mass spectroscopy) were provided by J. T. Baker (State of Mexico, Mexico) and Merck (California, USA), respectively. Commercial standards of polyphenolic compounds were obtained from Extrasynthese (Genay, France), Sigma-Aldrich (Steinheim, Germany), and Fluka (Dorset, U.K.). Filters (0.45 μm, HVLP04700) were obtained from Millipore (São Paulo, Brazil). Porcine enzymes used in in vitro gastrointestinal digestion and all reagents used for redox markers in cell culture were purchased from Sigma-Aldrich (Buenos Aires, Argentina). SnakeSkin dialysis bags with a molecular weight cut-off of 10 kDa and a width of 22 mm, and Hypersep SPE 500 mg/2.8 mL C18 cartridges were obtained from ThermoFisher SCIENTIFIC. Anaerobic atmosphere generation bags were purchased from Mitsubishi Gas Chemical (Tokyo, Japan). Dulbecco's modified Eagle medium was obtained from Sigma-Aldrich and fetal bovine serum from Natocor (Córdoba, Argentina). All other reagents were of analytical grade.

Total protein concentration was extracted from HeLa cells using a RIPA lysate (89901, Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was measured using a protein detection kit (A53227, Thermo Fisher Scientific, Waltham, MA, USA). A volume of 30 μg protein for each cell was transferred to a PVDF membrane (HVLP04700, Millipore, Billerica, MA, USA) using the SDS-PAGE method. After the transfer was completed, the membrane was soaked from bottom to top with TBS, then placed in an incubation box containing a 5% skimmed milk powder solution (37°C, 1 h), incubated with shaking at room temperature on a decolorization shake flask for 2 hours, and then incubated with phosphorylated (p)-Akt (1: 1000; 56 KD; ab38449; Abcam, Cambridge, UK), Akt (1: 500; 55 KD; ab8805), Bax (1: 1000; 21 KD; ab32503), Bcl-2 (1: 1000; 26 KD; ab59348), Wnt1 (1 μg/mL; 41 KD; ab85060), MMP-9 (1 μg/mL; 95 KD; ab73734), EGFR (1: 1000; 134 KD; ab52894), β-catenin (1: 1000; 92 KD; #9562; Cell signaling technology, Danvers, MA, USA), or β-actin (1: 1000; 45 KD; #4970) overnight at 4°C. The target band was incubated with a goat anti-rabbit IgG H&L (HRP) (1: 5000; ab205718; Abcam, Cambridge, UK) for 2 h. Finally, the signals were detected using SignalFir ECL reagent (#6883) and the gray value of the band was analyzed and calculated using ImageJ (version 5.0, Bio-Rad, Hercules, CA, USA) [17 (link)].