Anti cparp

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)].

Cell proteins were prepared using cell lysis buffer. Equal amounts of protein (50 mg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Merck Millipore) by electro-blotting. The membranes were blocked with 5% nonfat dry milk in TBST for 1 h, and then incubated with primary antibody anti-PU.1 (Abcam, Cat#:ab76543), anti-LC3B (Abcam, Cat#:ab48394), anti-p62 (Abcam, Cat#:ab56416), anti-caspase3 (Abcam, Cat#:ab13586), anti-c-caspase3 (Abcam, Cat#:ab2302), anti-PARP (Abcam, Cat#:ab32138), anti-c-PARP (Abcam, Cat#:ab32064), anti-ATG5 (Abcam, Cat#:ab227132), anti-ATG16L1 (Abcam, Cat#:ab188642) and anti-GAPDH (Abcam, Cat#:ab8245) overnight at 4 °C before subsequent incubation with second antibody (Cell Signaling Technology) for 1 h at 37 °C. Protein binds were visualized using enhanced chemiluminescence reagent (Pierce).