Caspase 1 activity assay kit

After infection with S. Typhimurium for 1 h and 3 h, the macrophages were collected. The caspase-1 activity in infected cell lysates was measured using the caspase-1 activity assay kit (Beyotime) and the protein concentrations were determined by the Bradford protein assay kit (Beyotime). One unit of caspase-1 vitality is definded as the amount of enzyme that will cleave 1.0 nmol of the substrate per hour at 37°C under saturated substrate concentration. Relative caspase-1 activity was expressed as the ratio of caspase-1 vitality to the total protein concentration (unit μg⁻¹)

The caspase-1 activity was assayed by using caspase-1 activity assay kit (Beyotime, China) according to the manufacturer's instructions. The absorbance was measured at a wavelength of 405 nm.

The caspase-1 activity was detected using a caspase-1 activity assay kit (C1102, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Briefly, 2 × 10⁶ HPAECs were harvested and lysed on ice for 15 min, centrifuged at 16,000×g for 15 min, and the supernatant was mixed with synthetic tetrapeptide Ac-YVAD-pNA and incubated at 37 °C for overnight. Finally, absorbance at 405 nm was recorded. The concentrations of total proteins were measured by a Bradford assay kit (P0006, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. The caspase-1 activity was calculated by the standard curve of pNA.

25HC was purchased from Sigma-Aldrich. Caspase-1 activity assay kit was purchased from Beyotime Institute of Biotechnology. Lipofectamine 3000 was purchased from Invitrogen. Annexin V-FITC and PI apoptosis detection kits were purchased from Keygen Biotech Company (Nanjing, China). Antibodies used in the present study were as follows: ALV-J envelope protein JE9-specific mouse monoclonal antibody (provided by Professor Qin Aijian of Yangzhou University), rabbit anti-LC3II antibody (NB100-2220SS, Novus Biologicals), rabbit anti-ATG5 antibody (12994, Cellsignal), mouse monoclonal anti-flag antibody (AP007M, Bioworld), rabbit anti- SQSTM1/p62 antibody (P0067, Sigma-Aldrich), and anti-myc antibody (A02060, Abbkine), rabbit anti-β-actin antibody (AF5003, Beyotime), goat anti-rabbit IgG (H+L) antibody (A21020, Abbkine), Goat anti-mouse IgG/HRP antibody (A25112, Abbkine).

The caspase-1 activity in various treatment groups was able to be detected by the caspase-1 activity assay kit (C1102, Beyotime, Shanghai) according to the attached detailed instructions. Briefly, after we collected the treated kidney tissue and HK-2 cells, the 100 μL reaction system was configured after successively adding the reagents from this kit according to the manufacturer's instructions. The samples were incubated at 37°C for 90 min. The absorbance at the wavelength of 405 nm was determined to assess the levels when the color change is obvious.

Apoptotic activities were analyzed by determining the amount of annexin V positive cells for apoptosis or the amount of pI-positive cells for necrosis using a flow cytometer (PA, Partec, Germany) (Zhao et al., 2011 (link)). The apoptosis rate was expressed as the percentage of apoptotic cells (%).
Caspase 1-like activities were measured using a Caspase 1 Activity Assay kit and Micro- Bradford assay kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. Total protein extracts (20 mg) were incubated consecutively for 2 h at 37°C with the synthetic tetrapeptide Ac-YVAD-pnitroaniline (pNA), during which, the addition of the substrate resulted in a signal caused by the caspase 1-dependent cleavage of the chromophore pNA from the labelled substrate. Caspase 1-like activities were measured at 405 nm. Enzymatic activities were expressed as the percentage of activity present in control extracts (%). Each measurement was conducted with three independent experiments.
VPE activities were measured following the methods with minor modifications described by Zhang et al. (2019a) (link).

A caspase-1 activity assay kit (Beyotime, China) was used to measure caspase-1 activity in cells according to the manufacturer's instructions. In brief, cells were lysed with ice-cold lysis buffer, and the protein concentration was detected by the Bradford method. The absorbance was measured at a wavelength of 405 nm with a TECAN Infinite 200 PRO series (Mannedorf, Switzerland).