Anti bax

Petri dishes were used to culture and treat MDA-MB-231 cells (2 × 10⁵ cells/mL) with AELE (261 and 522 μg/mL) for 24 h. The Qproteome mammalian protein prep kit (Qiagen, Hildren, Germany) was used to extract the proteins, which were then quantified based on the Lowry method. Proteins were prepared for separation by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (10%) as explained by El Zein et al. [41 (link)], and transferred to Polyvinylidene fluoride membranes for protein assessment, as previously described [42 (link)]. Membranes were incubated with primary antibodies (1:1000) anti-cytochrome c (Abcam, Cambridge, UK), anti-p21 (Cell Signaling, Danvers, MA), anti-Bax and anti-Bcl2 (Elabscience, Houston, TX, USA). The internal loading control was detected using anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). After washing, the secondary antibody (Bio-Rad, Hercules, CA, USA) was added at the recommended concentrations (2:5000). Blots were visualized on ChemiDoc machine (BioRad, Hercules, CA, USA) and relative expression of proteins bands was quantified using the ImageJ program [43 (link)].

After continuous infusion for eight hours, a small piece of the left ventricular myocardium was homogenized in a cold lysis buffer supplemented with protease inhibitors, and protein concentrations were evaluated using a bicinchoninic acid protein assay kit (Elabscience, Wuhan, China) with Tubulin as loading control. The samples of tissue lysis were heated to 95 °C for five minutes. Protein samples (40 µg) were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted onto polyvinylidene fluoride membranes overnight at 4 °C. Membranes were incubated with the appropriate primary antibody including anti-Bcl-2, anti-BAX, and anti-Tubulin, all from Elabscience, overnight at 4 °C. After washing with tris-buffered saline (TBST) three times, five minutes for each time, the secondary anti-rabbit immunoglobulin G antibody was then added and incubated for 30 minutes. After washing with TBST three times, proper amount of electrochemical luminescent substrate was added and incubated with the membranes in the dark. The results were scanned and processed by the ImageJ software.

Cells were seeded and incubated overnight at a density of 3 × 10⁵ cells/ml in 6-well plates, followed by treatment with increasing concentrations of AELE for 24 h. Total proteins were extracted, quantified, separated and transferred to polyvinylidene difluoride (PVDF) membranes,which were then blocked as previously stated by Abou Najem et al [37 (link)].
The membranes were incubated with primary antibodies anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Bax (Elabscience, Houston, TX, USA), anti-Bcl2 (Elabscience, Houston, TX, USA), and anti-cPARP (Abcam, Cambridge, UK), overnight in the fridge, with 2% skimmed dry milk in PBS with 0.05% Tween 20, at the manufacturer’s recommended concentrations: 1/1000 for anti-Bax, anti-Bcl2, anti-cPARP and 1/3000 for anti-actin. After washing, the membranes were incubated with anti-mouse secondary antibody (Bio-Rad, Hercules, CA, USA) at the recommended concentration (2:5000) for 1 h at room temperature. Another wash was performed, before imaging using Clarity™ Western ECL Substrate (Abcam, Cambridge, UK) on ChemiDoc machine (BioRad, Hercules, CA, USA). The ImageJ computer program was used to quantify the blot bands, in order to calculate the relative expression of proteins [37 (link)].