Ecl plus western blot detection system

Western immunoblotting analysis was performed to examine tyrosine phosphorylation in the sperm after incubation in coculture medium. Twenty micrograms of sperm protein were separated on 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and then transferred to a 0.45-mm hybond polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Germany). The membranes were blocked with 3% bovine serum albumin (BSA) for 1 h at 25°C. The membrane was subsequently incubated with the primary antibody (anti-phosphotyrosine antibody; Santa Cruz, USA; 1:500) overnight at 4°C. The membrane was washed with 1× tris-buffered saline with 0.1% Tween® 20 detergent (TBST) for 5 min 3 times and then incubated with a secondary antibody (goat anti-mouse immunoglobin G-horseradish peroxidase (IgG HRP) conjugated; Santa Cruz, USA; 1:1000) for 2 h at room temperature. The membrane was washed 3 times with 1× TBST for 5 min and visualized using an ECL plus Western blot detection system (Thermo Fisher Scientific Inc., Hampton, USA). The antigen-antibody reaction was detected using a Luminescent Image Analyzer (Imagequant LAS 4000, Sweden).

The mice were anesthetized and bilateral ovaries were collected. The tissue homogenates were centrifuged at 12,000 × g at 4 °C for 10 min. Bradford’s method was used to measure the protein concentration of supernatants [16 (link)]. The total proteins were subjected to electrophoresis in 8% SDS-PAGE gels and transferred onto a PVDF membrane. Subsequently, membranes were probed overnight at 4 °C with anti-FSHR (1:100), anti-AMH (1:1000) and anti-GAPDH (1:800; Goodhere, China) polyclonal antibodies. At room temperature, the horseradish peroxidase-conjugated secondary antibody (1:5000) was incubated on the membrane for 1 hour. The ECL Plus western blot detection system (Thermo Scientific) was used to detect the immunoreactive signals.

BMDMs or lungs were harvested at designated time points and homogenized in PBS containing 0.1% Triton X-100 (phosphatase and protease inhibitor cocktail added). After centrifugation the supernatants were used for immunoblotting. Total protein content in the supernatant was measured using a BCA protein assay kit (Thermofisher, NY) to ensure that equal amounts of proteins were loaded onto 10% SDS-PAGE gels. Proteins were transferred to polyvinylidene fluoride membrane according to the protocol provided by Bio-Rad. Appropriate primary antibodies against mouse NLRP6 (Sigma, MO), phospho-MLKL (Abcam, MA), RIP3, RIP1, P47^phox, P67^phox, gp91^phox, phospho-p38 MAPK, phospho-JNK, phospho-Stat3, caspase-8, GAPDH (Cell Signaling, MA), caspase-1 (Adipogen), and gasdermin-D (Santa Cruz, CA) were added to the membrane and incubated overnight at 4⁰ C. Appropriate secondary antibodies were used, and the films were developed using ECL plus western blot detection system (ThermoFisher, NY). IL-1β, TNF-α, IFN-γ, IL-1α, and IL-6 were measured in BALF supernatants by ELISA according to the manufacturer’s protocol (eBioscience, CA).