Odyssey infrared scanning system

Proteins were isolated with RIPA lysis buffer (Beyotime, Jiangsu, China) after 48h of transfection. Protein lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred to nitrocellulose membrane (Beyotime, Jiangsu, China). nitrocellulose membrane with proteins were immunoblotted overnight at 4°C with primary antibodies: anti-PCNA (Proteintech, USA), anti-Wnt7b (Proteintech, USA), β-catenin (Proteintech, USA), Gsk-3β (Proteintech, USA), cyclin D1 (Abcam, USA), C-myc (Wanlei, China). Subsequently, the membranes were incubated in secondary antibodies for 1h at room temperature. Dilutions of all antibodies used in this study were 1:1000. Odyssey Infrared scanning system (Li-Cor, Lincoln, NE, USA) was used to visualize protein bands.

Total proteins from the colonic tissues and cells were extracted using a standard extraction reagent supplemented with the protease inhibitor (KANGCHEN; Shanghai, China). Protein concentrations were calculated through the BCA kit (Beyotime, China), according to the manufacturer's instructions. Equal amounts of proteins (30 μg) were subjected to the SDS-PAGE gel and then transferred onto a polyvinylidene fluoride (PVDF; Millipore, USA) membrane. The membranes were blocked with 5% skim milk in PBS at room temperature for 1 h, followed by the incubation with the primary antibodies against rabbit anti-Akt (1 : 1,000), rabbit anti-p-Akt (1 : 2,000), rabbit anti-IKK (1 : 1,000), rabbit anti-NF-κB p65 (1 : 1,000), rabbit anti-p-NF-κB p65 (1 : 1,000), rabbit anti-caspase-1 P20 (1 : 1,000), rabbit anti-Gasdermin N (1 : 1,000), rabbit anti-PHLPP2 (1 : 2,000), and β-actin (1 : 2,000) for 24 h at 4°C. Subsequently, the blots were washed with TBST three times (10 min for each wash) and incubated with anti-rabbit IgG (1 : 10,000) for 1 h. Finally, immunoreactive bands were visualized with an Odyssey infrared scanning system (LI-COR Biosciences, USA) and quantified using ImageJ software.

Total proteins were extracted by RIPA lysis buffer (Beyotime, China) together with PMSF (Beyotime, China). The concentration of total protein was determined by BCA protein assay kit (Beyotime, China). After being separated by 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels, proteins lysates were transferred to nitrocellulose membranes (Beyotime, China). The membranes were blocked with 5% non-fatty milk for 1 h at room temperature and then immunoblotted at 4 °C overnight with primary antibodies: anti-WBP2 (1:1000, Proteintech, USA), anti-AKT (1:1000, Proteintech, USA), anti-p-AKT (1:1000, Abcam, USA) and anti-β-actin (1:10,000, Abclonal, China). After incubated with diluted secondary antibodies for 1 h at room temperature, the bands were scanned and analyzed by Odyssey Infrared scanning system (LI-COR Biosciences, USA). Original western blots were shown in Fig. S1.

Total protein was extracted with the procedures as described [41 ]. Briefly, Protein samples (80-100 μg) were separated by 10% acrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membrane. The membranes were blocked with 5% defatted milk for 2-3 h at room temperature on a rocker and then incubated with primary antibodies β₂-AR (Abcam, Cambridge, MA, UK) and beta-actin (Proteintech, Wuhan, China) at 4°C overnight. After washing with PBS-T (PBS containing 0.5% Tween 20) for 3 times, the membranes were incubated with infrared fluorescent dye-labeled secondary antibody (LI-COR Biosciences, Lincoln, USA) for 50 min at room temperature away from light. Western blot bands were acquired using Odyssey infrared scanning system (LI-COR Biosciences, Lincoln, USA) and analyzed using Image Studio Ver 4.0 software.

For western blotting, cells were lysed in PBS containing 2% Triton X-100 and Pierce protease and phosphatase inhibitor tablets (Thermo Fisher Scientific). The protein concentration was determined with a BCA assay kit (Thermo Fisher Scientific), and equal amounts were resolved on NuPAGE 4–12% precast gels (Invitrogen). Proteins were transferred from gels onto PVDF membrane (Immobilon-FL, pore size 0.45 μm, Millipore, catalogue number IPFL00010) for immunoblotting. Transfer was performed in transfer buffer (25 mM Tris, 192 mM glycine and 10% methanol) at 100 V for 70 min. Membranes were blocked in 5% milk in PBS containing 0.1% Tween 20 (PBS-T) prior to incubation with the appropriate primary antibodies and fluorescently labelled secondary antibodies. Detection and quantification were carried out using an Odyssey infrared scanning system (LI-COR Biosciences).