Anti caspase 7

Cell lysates were prepared by resuspending cell pellets in 1× Laemmli sample buffer containing 5% β-mercaptoethanol. Protein from lung tissues were extracted using IPH lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% NP40) containing protease inhibitor cocktail (Roche). The protein lysates were separated by SDS–PAGE and then electrotransferred to nitrocellulose membranes (Millipore Corp., Bradford, MA, USA). Detection of specific proteins was carried out with enhanced chemiluminescence reagents following the manufacturer’s instructions (P90720, Millipore Corporation, MA, USA). The primary antibodies used for western blotting were as follows: anti-Caspase-9 (#9508), anti-Caspase-8 (#9746), anti-Caspase-7 (#8438), anti-Caspase-3 (#9664), anti-Bim (#2933), anti-Bak (#3814), anti-Bcl-xL (#2762), anti-p-Jak2 (#3771), anti-Jak2 (#3230), and anti-p-STAT3^Y705 (#9145), which were purchased from Cell Signaling Technology. Anti-CDK2 (sc-6248), anti-p53 (sc-126), anti-STAT3 (sc-8019), anti-PARP-1 (sc-74470), anti-Cyclin D1 (sc-8396), and anti-β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology. An anti-p21 (ab7960), anti-p16 (ab108349), anti-IL-6 (ab6672), and anti-TNF-α (ab6671) antibodies were purchased from Abcam. Densitometry analyses were performed using ImageJ software (National Institutes of Health, NIH).

Western blot analysis was carried out as described by Misiura et al. [30 (link)]. The membranes were incubated with primary antibodies diluted 1000 times in 5% bovine serum albumin (Sigma Aldrich, Saint Louis, MO, USA) in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6). Anti-PARP, anti-AMPK and anti-caspase-7 and anti-GAPDH, were purchased from Cell Signaling Technology, Danvers, MA, USA; anti-PRODH/POX from St John’s Laboratory, London, UK), followed by incubation with alkaline phosphatase-linked goat anti-rabbit or anti-mouse antibodies (dilution: 1:10,000 in 5% non-fat dried milk (Santa Cruz Biotechnology, Dallas, TX, USA) in TBS-T; Sigma Aldrich, Saint Louis, MO, USA). The bands’ intensities were semi-quantitatively measured in ImageJ software (https://imagej.nih.gov/ij/, accessed on 27 October 2021). All experiments were run at least in triplicates.

Cells were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 1 mM sodium vanadate, 1 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 0.1 mg/ml pepstatin, 0.1 mg/ml leupeptin, and 0.1 mg/ml aprotinin). The protein concentration was determined by using a bicinchoninic acid protein assay. Protein lysates (40 μg) were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride membranes (PVDF; Bio-Rad, Hercules, CA, USA), and blotted with different primary antibodies [anti-IL-18; Abcam, Cambridge, UK; anti-GSK-3β, anti-phosphorylated GSK-3β (p-GSK-3β), anti-caspase-3, anti-cleaved caspase-3, anti-caspase-7, anti-cleaved caspase-7; all from Cell Signaling Technology, Boston, MA, USA] overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies and visualized with an ECL reagent (GE Healthcare, London, UK).