Anti caspase 3

Total protein from liver tissue was extracted and quantified with a bicinchoninic acid protein concentration assay kit (Thermo Scientific) and then separated by 12% SDS/PAGE with a Bio-Rad electrophoresis system. Blotted membranes were incubated for 1 h with blocking solution (Tris-buffered saline/0.1% Tween 20, TBST) containing 5% skim milk (w/v) at room temperature. Subsequently, the membrane was incubated overnight at 4 °C or 1 h at room temperature with primary antibody [anti-granzyme B (catalog: A2557), anti-perforin (catalog: A0093), TRAIL (catalog: A12064), and anti-Caspase-3 (catalog: A19654) polyclonal antibody from Abclonal (Wuhan, China); and anti-Caspase-8 polyclonal antibody (catalog: 13423-1-AP) and anti-GAPDH monoclonal antibody (catalog: 60004-1-Ig) from Proteintech (Wuhan, China)]. After being washed, the membrane was incubated 1 h with 1:10,000 diluted HRP-conjugated polyclonal goat anti-rabbit IgG (H+L) or HRP-conjugated polyclonal goat anti-mouse IgG (H+L) antibodies (Proteintech, China). The membranes were washed, and the immunoblot was then developed with an ECL chemiluminescence detection kit (Thermo Scientific) according to the manufacturer’s instructions. To normalize for protein loading, GAPDH was used, and the protein expression levels were calculated relative to the value of GAPDH.

Paraffin-embedded mouse kidney sections (3 µm thickness) were prepared by routine procedures. The sections were stained with periodic acid-Schiff (PAS) reagents by standard protocol. For immunohistochemical staining, kidney sections were deparaffinized with xylene, and then gradually rehydrated in ethanol. Hydrogen peroxide (3%) was used to eliminate endogenous peroxidase. Antigen retrieval was performed by microwave using the heat mediated antigen retrieval technique. After blocking with 1% donkey serum for 1 h, the sections were incubated with primary antibodies overnight at 4°C and subsequently washed and incubated with secondary antibodies for 1 h at room temperature. The sections were visualized by using AEC substrate Kit under an Olympus light microscope. The primary antibodies were as follows: anti-Coronavirus nucleocapsid (sc-66012; Santa Cruze Biotechnology), anti-KIM-1 (AF1817; R&D Systems), anti-p53 (A0263; Abclonal), anti-caspase-3 (A2156; Abclonal).

Total protein was extracted by lysing cells in lysis buffer (20 mM HEPES, pH 7.4, 20 mM NaCl, 10% glycerol, and 1% Triton X-100). Colonic membrane proteins were prepared using a Membrane Protein Extraction Kit (Biovision, Milpitas, CA, USA) and soluble proteins were isolated from colon homogenates using methanol and chloroform (28 (link)). All protein samples were subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Merck KGaA, Darmstadt, Germany). Immunoblotting was performed with the following primary antibodies: anti-tmTNF-α (home-made) (31 (link)), anti-TNF-α (Cat# 3707s), anti-PARP (Cat# 9532s), anti-cleaved caspase 3 (Asp175) (Cat# 9661s) from Cell Signaling Technology (Danvers, MA, USA), anti-IκB-α (Santa Cruz, CA, USA, Cat# sc-1643), anti-p65 (Cat# A19653), anti-p-p65 (Cat# AP0475), anti-caspase 3 (Cat# A17900), anti-Na⁺/K⁺ ATPase (Cat# A12405), and anti-β-actin (Cat# AC026) from Abclonal (Wuhan, China). HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA, Cat# 7074) were subsequently applied to the membrane. Bands were visualized using an enhanced chemiluminescence system (ECL; TIANGEN, Beijing, China).