Phospho pi3k

Immunohistochemistry was performed using an Immunohistochemistry Kit (Boster, Wuhan, China). The preparation of paraffin sections of eyeballs was the same as for HE staining. After the paraffin sections were dewaxed, they were heated with 3% citric acid solution for antigen repair, and a goat serum from the kit was used to block the non-specific antigen before the primary antibody incubation (phospho-PI3K: dilution 1:200; phospho-Akt 1/2/3: dilution 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hours at 37°C, followed by 30 minutes’ incubation with the goat anti-rabbit IgG (dilution 1:200; Zhongshan Jinqiao, Beijing, China) at 37°C. Then DAB reagent was used to develop the color under the microscope for 40 seconds. After re-staining with hematoxylin and gradient dehydration with alcohol, the film was sealed with neutral gum and photographed using an optical microscope (Nikon).

The human colon cancer cell line LoVo was obtained from the American Tissue Culture Collection (ATCC) (Rockville, MD, United States). LoVo cells were established from metastatic nodules that were resected from a 56-year-old colon adenocarcinoma patient.
We utilized antibodies against the following proteins: phospho-PI3K, phospho-Akt, COX-2, phospho-GSK3β, β-catenin, LEF-1, HADAC-1 (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, United States), and TCF-4 (Cell Signaling Technology, Inc. Beverly, MA, United States). α-tubulin and β-actin (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, United States) were used as loading controls. The following horseradish peroxidase-conjugated antibodies were purchased from Santa Cruz Biotechnology, Inc. (CA, United States): goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG. Nude mice were purchased from the National Laboratory Animal Center (NLAC).

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.