Anti caspase 8

Carvedilol was purchased from GNP company (6th of October, Giza, Egypt). Cadmium chloride (CdCl₂) and ACET were purchased from Sigma Aldrich (St. Louis, MO, USA). Rat tumor necrosis factor-alpha (TNF-α; CAT# E-EL-R2856), troponin-I (CAT# E-CL-R0721), and (IL-6; CAT# E-EL-R0015) ELISA kits were purchased from ELABSCIENCE (Wuhan, China). Primers for Sirtuin 1 (SIRT1), Forkhead box O3 (FOXO3), KEAP-1, Nrf2, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes were synthesized by Vivantis Technologies (Malaysia). Anti-RIPK1 (CAT# PA5-20811), anti-RIPK3 (CAT# PA5-19956), anti-MLKL (CAT# PA5-115578), and anti-caspase-8 (CAT# PA5-20118) were purchased from ThermoFisher Scientific (USA). Anti-iNOS (CAT# sc-7271), IκB (CAT# sc-1643), and anti-TLR4 (CAT# sc-293072) were purchased from Santa Cruz (USA). Anti-NF-κB (CAT# abx012874) was purchased from Abbexa (Germany). Anti-NADPH oxidase (CAT# E-AB-70215) and anti-β-actin (CAT# E-AB-20031) were purchased from ELABSCIENCE (China). Anti-Nrf2 (CAT# YPA1865) and anti-HO-1 (CAT# YPA1919) were purchased from Biospes (China). ACET and CV dissolved in 0.5% carboxymethyl cellulose (CMC).

Protein lysates were sonicated and subjected to Tris-glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to nitrocellulose membranes. Membranes were then blocked for 1 h with 5% nonfat milk, incubated with primary antibody overnight and secondary antibody for 1 h. The primary antibodies included: anti-Id3 (C-20; Santa Cruz Biotechnology), anti-GAPDH (EMD; Millipore, Billerica, MA), anti-active caspase-3 (Enzo Life Sciences, Inc., Farmingdale, NY), anti-caspase-8 (Thermo Scientific Rockford, IL), and anti-Elk-1 (I-20; Santa Cruz Biotechnology).

The levels of proteins engaged in the apoptotic process (caspase 8, procaspase 9, and procaspase 3) and autophagy, i.e., LC3-II, were tested. A sample was separated with SDS-PAGE and transferred to a PVDF membrane (Amersham, GE Healthcare Life Sciences, Freiburg, Germany) using vertical electrophoresis apparatus Mini-PROTEAN^® 3 Cell (BioRad, Hercules, CA, USA). The proteins were immunoblotted with the primary monoclonal antibodies: anti-caspase 8, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Thermo Fisher Scientific, Waltham, MA, USA), and anti-caspase 3 and 9 (Cell Signaling, Leiden, The Netherlands). GAPDH was amplified as an internal control. Protein bands were visualized with a Bio Imaging System (DNR Lumi BIS, Jerusalem, Israel) using the fluorescent method of a WesternDot kit (Thermo Fisher Scientific). Protein bands were characterized using the ImagineR (Sun Microsystems, Santa Clara, CA, USA) analysis software [50 (link)].