E ab 1001

As in our previous study, the liver samples were lysed using a radioimmunoprecipitation assay lysis buffer. After protein content analysis, 40 µg isolated proteins were separated using 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.45-μm polyvinylidene difluoride membranes (Merck, Darmstadt, Germany). The membranes were then blocked with 5% bovine serum albumin at 4 °C for 4 h and then incubated with primary antibodies, including CAT (#ab209211, 1:2000 dilution), Nrf2 (#ab92946, 1:1000 dilution), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#ab181602, 1:1000 dilution) (Abcam, Cambridge, MA, USA), heme oxygenase 1 (HO-1, #A19062, 1:1000 dilution), and superoxide dismutase 1 (SOD-1, #A12537, 1:1000 dilution) (Abclonal, Wuhan, China) overnight at 4 °C. The horseradish peroxidase (HRP)-conjugated secondary antibodies (#E-AB-1001 and #E-AB-1003) (Elabscience, Wuhan, China) were used to incubate with the membranes at 4 °C for 4 h. Protein bands were developed using an enhanced chemiluminescence detection kit (Merck, Darmstadt, Germany) and visualized using a Tanon 5200 gel imaging system (Tanon Science & Technology Co., Ltd., Shanghai, China). The pixel density was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA) [23 (link)].

The colorectum, heart, liver, spleen, kidney, and lung specimens collected from Apc^Min/+ mice were fixed in 4% paraformaldehyde for 48 h. Samples were embedded in paraffin, sliced into 5 μm sections, and stained with hematoxylin and eosin (H&E), as described in a previous study (Elsherbiny et al., 2017 (link)).
Other sections of the colorectum (especially the colorectal tumor portion) and spleen were blocked with 5% bovine serum albumin (Gen-view Scientific, Galveston, TX, United States ) for 4 h and incubated with primary antibodies including CD4 (25229S, 1:200 dilution) and CD8 (98941S, 1:800 dilution) (Cell Signaling Technology, Danvers, MA, United States ), IFN-γ (PA5-95560, 1:1000 dilution) and IL-4 (PA5-25165, 1:50 dilution) (Invitrogen, Carlsbad, CA, United States ) at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibodies (E-AB-1003 or E-AB-1001) (Elabscience, Wuhan, China) at 4°C for 2 h. Color development was performed using the Metal Enhan DAB Substrate Kit (34,065, Thermofisher, Carlsbad, CA, United States ), and hematoxylin was used for counterstaining. All the slides were observed under a light microscope (Olympus Corporation, Tokyo, Japan). ImageJ software (National Institutes of Health, Bethesda, MD) was used to quantify the pixel density for the semi-quantitative densitometric analysis of protein expressions.

RWPE-1 and PC-3 cell extracts were obtained by cultivating these cells, separately, in 6-well plates. Upon confluence, the monolayers were washed and a protein extraction buffer was added NP40 (Invitrogen™, with 1% proteinase inhibitor). Total protein in cell extracts were quantified using a spectrophotometer at 260/280 nm with the Pierce™ BCA Protein Assay Kit. Cell extracts (35 µg/lane) were boiled for 5 min in SDS protein sample buffer and subjected to 12% SDS PAGE at 120V for 90 min.
Proteins were transferred to a nitrocellulose membrane using the Amersham Biosciences^® Mighty Small Transphor at 0.4 A for 2 hours in transfer buffer (250 mM Tris and 1,92 M glicin) at pH 8.5. The blotting membranes were blocked using blocking buffer solution (5% non-fat dry milk in TBST) containing 50 mM Tris, 150 mM NaCl and 0,1% (m/v) Tween 20 for 1 h at room temperature. The purified mAbs 2-7A50 e 2-5C11 (primary antibodies) were incubated overnight at 4°C (1:50). The secondary antibody used was a Goat anti-mouse IgG peroxidase/HRP conjugated (Elabscience™, E-AB-1001) (1:5,000). Antibody binding was detected using the Enhanced Chemiluminescence System (ECL). Images were registered using Chemidoc XRS (Bio-Rad™) equipment.

Cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, China) containing protease inhibitor cocktail. BCA protein quantification kit (Vazame, China) was used to detect the protein concentration. Equal amounts of protein were separated by SDS-PAGE and then transferred to PVDF membrane. After blocking with 5% BSA for 1 h, the membranes were incubated overnight at 4 °C with the indicated primary antibodies, followed by incubation with HRP-labeled secondary antibody for 1 h at room temperature. Primary antibodies were purchased from Cell Signaling Technology (E-cadherin, 1:1000, #3195; N-Cadherin, 1:1000, #13,116; Snail, 1:1000, #3879; MMP-2, 1:1000, #40,994; MMP-9, 1:1000, #13,667; Phospho-Akt, 1:1000, #4060; Akt, 1:1000, #4691; PI3K, 1:1000, #3011), Bioss (Phospho-PI3KCA, 1:1000, bs-5570R), Proteintech (TEAD4, 1:2000, 12,418–1-AP; Vimentin, 1:1000, 10,366–1-AP), and ImmunoWay (GAPDH, 1:10,000, YM3209). Secondary antibodies were purchased from Elabscience (Goat Anti-Rabbit IgG (H + L), 1:10,000, E-AB-1003; Goat Anti-Mouse IgG(H + L), 1:10,000, E-AB-1001).

Total protein was extracted from cells in each group using RIPA lysis buffer (P0013D; Beyotime, China). The protein content was evaluated using Pierce™ Rapid Gold BCA Protein Assay Kit (A53225; Thermo Fisher Scientific). Next, the protein was separated on 10% SDS-PAGE gel, transferred onto PVDF membranes (IPVH00010; Millipore, Burlington, MA, USA), and blocked with 5% skimmed milk under room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with anti-PTPL1 goat polyclonal primary antibody (1:1000; AF3577; R&D) and anti-GAPDH mouse monoclonal primary antibody (1:10000; ab8245; Abcam, Waltham, MA, USA). Then the membranes were washed in Tris-Buffered Saline and Tween (TBST) and incubated with HRP-conjugated donkey anti-goat secondary antibody (1:3000; E-AB-1050; Elabscience, Houston, TX, USA) or HRP-conjugated goat anti-mouse secondary antibody (1:3000; E-AB-1001; Elabscience) at room temperature for 1 h. After washing three times in TBST, images were acquired with an Electro-Chemi-Luminescence (ECL) chemiluminescence kit (P0018M; Biyuntian Bio, Shanghai, China). Signal intensities of bands were quantified using Image J software.