2.8. Immunoblotting of apoptosis markers

Cells were treated with 15 µg/ml MeCc for 24 h prior to protein extraction. Collected cells were extracted by a protein extraction buffer composed of 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM PMSF, 0.1% NP-40 and 1x complete protease inhibitor (Roche Diagnostics GmbH, Mannheim, Germany) using a stick “pellet pestle blue” (Sigma Aldrich, St Louis, MO, USA). The total proteins were quantified using Bradford assay method (Bio-Rad® Laboratories GmbH, Munich, Germany). Forty µg protein of each treatment were loaded into 7–12% SDS-PAGE gels depending on the size of target protein. Then the gels were transferred to a PVDF membrane using a semidry transfer blot system. The membranes were blocked using 5% non-fat milk diluted in PBS buffer 0.1% Tween-20 (PBS-T) and subjected to immunodetection by the anti-Caspase-3 polyclonal antibody (ab13847; Abcam UK), anti-p53 monoclonal antibody (PAb 240; ab26; Abcam UK), anti-Bcl-2 monoclonal antibody (E17; ab32124; Abcam UK) and anti-Bax monoclonal antibody (E63, ab32503; Abcam UK). anti-β-Actin monoclonal antibody (SP124; ab115777; Abcam UK) was used as an internal control, whereas anti-Mouse and anti-Rabbit were used as secondary antibodies. Dilution rates were either 1:1000 or 1:2000 dependging on the respective antibody used (El-Hallouty et al., 2020, Aboul-Soud et al., 2020).