Pro il 1β

Standard Western blot analysis was performed on the duodenum, jejunum, and ileum tissues of birds. Briefly, tissues were homogenized with protein lysis buffer. Protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific) as per the manufacturer's instructions and using a multimode microplate reader for spectroscopic measurements. About 30 μg proteins were mixed with 3 μL loading buffer and subjected to 10% SDS-PAGE electrophoresis followed by blotting onto a polyvinylidene difluoride membrane. Membranes were immersed in blocking solution (3% BSA in tris buffered saline tween) for 2 h and then immunoblotted with primary antibodies (Hsp70, TLR4, NF-κBp65, caspase-1, pro-IL-1β, IL-1β, IL-6, TNF-α, and β-actin; Cell Signaling Technology, Danvers, MA) diluted with 3% BSA for 2 h at room temperature. The membranes were washed with tris buffered saline tween buffer 3 times followed by 1 h incubation with HRP-conjugated secondary anti-mouse IgG from goat (or anti-rabbit IgG). And the gray level of the protein expression was analyzed by using the software, ImageJ.

WBs were performed
as described previously.^{60 (link)} Tissue homogenates
and cells were prepared in lysis buffer (Beyotime, Shanghai, China),
consisting of 1 nM phenylmethanesulfonyl fluoride (Beyotime, Shanghai,
China) to extract protein, and the protein concentration was detected
using an BCA protein detection kit. An equal amount of protein solubilization
was added to the band and separated by 10% sodium dodecyl sulfate
polyacrylamide gel. Then the isolated protein was transferred to a
polyvinylidene fluoride membrane (Millipore Corporation, Darmstadt,
GER). After incubation in 5% skimmed milk containing 0.1% TBST for
1 h, the membrane was incubated with primary antibodies against mouse
PI3K(A1520, Santa Cruz Biotechnology, CA, USA), phospho-PI3K(D1718,
Santa Cruz Biotechnology, CA, USA), p38(C0218, Santa Cruz Biotechnology,
CA, USA), and phospho-p38 antibody (I1719, Santa Cruz Biotechnology,
CA, USA), NLRP3(A27381510, Adipogen, Liestal, SUI), NF-κB p65(#F2912,
Santa Cruz Biotechnology, California, USA), p-NF-κB p65 (16,
Cell Signaling Technology, Boston, MA, USA), and Pro-IL-1β (#12242,
Cell Signaling Technology, Boston, MA, USA) or GAPDH (BC004109, Proteintech,
PA, USA) in 1:1000 dilution overnight at 4 °C. Then, the secondary
antibodies were added, and the membrane was incubated at room temperature
for 1 h. In each sample, the target protein expression level was normalized
to GAPDH.