Bradford assay kit

The samples (10–12 workers) treated with LC₃₀ and control were weighed before homogenization. The fire ant workers were uniformed at room temperature with ice-cold 0.05 M sodium phosphate buffer (pH 7.3). At 4 °C, homogenized RIFA samples were centrifugated at 12,000 rpm for 10 min. The supernatant was extracted, transferred into new tubes, and centrifuged at 12,000 rpm for 15 min. The last supernatants were used for various enzyme analyses. The activity of the detox enzyme (AChE, CarE, GST) was identified by commercially available kits bought from the Bioengineering Research Institute of Nanjing Jiancheng, Nanjing City, China. The manufacturer’s instructions were followed. Bio-Rad spectrophotometer (iMark) (OSAKA, Japan) used at absorbance light of 412 and 450 nm wavelength (according to the manufacturer’s protocol). Additionally, the protein concentration in the sample was measured using a Bradford assay kit acquired from Beyotime, Shanghai, China, and standardized with BSA (bovine serum albumin) [22 (link)].

The cells were collected and washed twice with ice-cold PBS, and were subsequently lyzed in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 274 and 0.1% SDS) supplemented with protease and phosphatase inhibitors (1 mM PMSF, 5 μg/ml 275 leupeptin, 2 μg/ml aprotinin, 1 mM EDTA, 10 mM NaF, and 1 mM NaVO₄). The lysates were centrifuged for 10 min at 10,000 g at 4°C. The supernatants were collected, and the protein concentration was determined using Bradford assay kit (Beyotime, China). The protein was separated on 12% SDS-PAGE, which was then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, United States). The PVDF membranes were blocked by 5% nonfat milk, and was incubated with the indicated primary-antibody solution at 4°C overnight, followed by incubation with peroxidase-conjugated secondary antibodies for 1.5 h. The resulting bands were tested using chemiluminescent reagents on a ChemiDoc XRS system (Bio-Rad, United States). Antibodies against β-actin (1:2000), AKT (1:1000) and AKT (phospho-Ser473, 1:1000) were purchased from Bioworld (China). Antibodies against GLUT1 (1:1000) and G6PD (1:1000) were purchased from Cell Signaling Technology (United States).

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.