Clarity western ecl substrate

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)].

KG-1 cells were plated in a 6-well plate at a density of 10⁶ cells/mL before treatment with two increasing concentrations of ASEE for 24 h (38 and 76 μg/mL). Control cells were treated with RPMI media. Total proteins were extracted using the Q-proteome Mammalian Protein kit (Qiagen, Hilden, Germany) and quantified using the DC (Detergent Compatible) protein assay (Bio-Rad). Proteins were separated by SDS-PAGE; transferred to PVDF (Polyvinylidene fluoride) membranes which were blocked with 5% skimmed milk; and then incubated with primary antibodies anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Cytochrome-c, anti-cleaved PARP-1, anti-Bax, anti-Bcl2, and anti-caspase-9 (Abcam, Cambridge, UK), anti-p53, and anti-caspase-8 (Elabscience, Houston, TX, USA) at the manufacturer’s recommended concentrations. After washing and incubation with a secondary antibody (Bio-Rad, Hercules, CA, USA), membranes were washed and image development was done using the Clarity™ Western ECL Substrate (Abcam, Cambridge, UK) on the ChemiDoc machine (BioRad, Hercules, CA, USA). Blot bands were quantified using the ImageJ computer program to calculate the relative expression of proteins.

Monomac-1 cells were plated in 6-well plates at a density of 5 × 10⁵ cells/mL before treatment with two increasing concentrations of MMLE for 24 h. The concentrations used were the closest to the IC50. Total proteins were extracted using the Qproteome mammalian protein prep kit (Qiagen, Hilden, Germany) and quantified using the Lowry method. Proteins were then separated by SDS-PAGE (10%) and transferred to PVDF membranes that were blocked with 5% skimmed milk, then incubated with primary antibodies: anti-β-actin (Santa Cruz Biotechnology, Dallas, Tx, USA), anti-cytochrome-c and anti-cleaved poly(ADP-ribose) polymerase (PARP) (Abcam), anti-Bax and anti-Bcl2 (Elabscience, Houston, TX, USA). β-actin was used as a loading control. Membranes were then washed and incubated with a secondary antibody (Bio-Rad, Irvine, CA, USA) followed by exposure for image development using Clarity™ Western ECL substrate (Abcam) on a ChemiDoc machine (Bio-Rad). Quantification using the ImageJ program allowed us to calculate the relative expression of proteins, as compared to the loading control.