Ecl reagent

Total protein was extracted with RIPA (radio immunoprecipitation assay) lysate. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) 50 μg of protein samples were subjected to SDS-PAGE and transferred onto PVDF membranes (Solarbio, Beijing, China). The membrane was blocked with 5% nonfat dry milk (Solarbio, Beijing, China) for 1.5 h, then incubated with specific primary antibodies at 4 °C overnight. Following incubation with HRP-conjugated secondary anti-rabbit antibody (7074, Cell Signaling Technology, Danvers, MA, USA), membranes were examined with ECL reagent (KF005, Affinity Biosciences, Melbourne, Australia) and visualized on ChemiDocTM XRS + Imaging System (BioRad, Hercules, CA, USA). ImageJ software (U.S. National Institute of Health, Bethesda, MD, USA), was applied to detect and analyze the gray values of protein bands on the membrane. The following primary antibodies: transforming growth factor (TGF)-β, interleukin (IL)-10, tumor necrosis factor (TNF)-α, interleukin(IL)-6, NADPH oxidase 2 (NOX2), P22, arginase-1 (Arg-1), C–C motif chemokine ligand 3 (CCL3), interleukin(IL)-1β, monocyte chemoattractant protein-1 (MCP-1), C–X3–C motif chemokine ligand 1 (CX3CL1), and β-actin. All the above antibodies purchased form Proteintech, Beijing, China.

Ovarian tissues and cells were lysed in RIPA buffer and the protein extracts were then separated by SDS-PAGE. The separated proteins were then transferred onto PVDF membranes, followed by blocking with 5% skim milk. The membranes were first incubated with primary antibodies against VEGFA (sc-7269; 1:500), PTX3 (sc-373951; 1:200), and GAPDH (sc-365062; 1:200; all from Santa Cruz Biotechnology, Shanghai, China) at 4 °C overnight and then with the corresponding HRP-conjugated goat anti-mouse IgG secondary antibody (31,430; 1:20,000; Thermo Fisher Scientific, Shanghai, China) for 1 h at room temperature. The blots were visualized using an ECL reagent (Affinity, Changzhou, China) and were semi-quantified using ImageJ software.

Total cellular protein was extracted with RIPA lysis buffer, and protein concentration of lysates was measured by BCA Protein Assay Kit (Thermo, Waltham, USA). The protein samples were stored at −80°C for further analysis. Subsequently, equal amounts of proteins were separated by 10% SDS-PAGE gel and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA). After blocking in 5% non-fat milk in TBST buffer for 1 h at room temperature, the membranes were incubated with three primary antibodies, respectively, including mouse anti-vinculin antibody (Sigma; V9131; 1:1500), mouse anti-Col-I antibody (Abcam; ab88147; 1:2000), mouse anti-α-SMA antibody (Sigma; A5228; 1:2000), or mouse anti-β-actin antibody (Affinity; T0022; 1:3000) at 4°C overnight, followed by incubation with HRP-linked goat anti-mouse IgG (H + L) (Affinity; S0002; 1:3000) at 37°C for 1 h. After washing the membranes with TBST, the band signals were visualized by using Affinity® ECL Reagent. Finally, the membranes were exposed and analyzed by using Luminescent Image Analyzer (Amersham, Uppsala, Sweden). The results of each blot were normalized against β-actin protein expression.

The cells were lysed in RIPA solution (Beyotime, China) with a protease inhibitor PMSF (Solarbio, China). The isolated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane (Millipore, USA). The membrane was washed in 5% milk for 1 h and then incubated with a primary antibody overnight. The antibodies including anti-COL2A1 (28459-1-AP), anti-ACAN (13880-1-AP), anti-MMP3 (17873-1-AP), anti-MMP13 (18165-1-AP), anti-Alix (12422-1-AP), anti-CD63 (25682-1-AP), anti-Notch1 (20687-1-AP), anti-Hey1 (19929-1-AP), and anti-Hey2 (10597-1-AP) were purchased from Proteintech company (Wuhan, China). The antibodies including anti-Vasorin (ab156868), anti-Calnexin (ab133615), and anti-GAPDH (ab8245) were purchased from Abcam company (Cambridge, UK). After incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, the bands were visualized using an ECL Reagent (Affinity Biosciences, USA).

A protein extraction kit (KeyGEN Biotech, Nanjing, China) was used to extract total proteins from colon cancer cells. Then, protein concentrations were quantified using a BCA Protein Assay kit (KeyGEN Biotech, Nanjing, China). After boiling for 5 min with loading buffer, each sample containing an equivalent amount of protein (20 μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk powder for 1 h at room temperature, the PVDF membranes were incubated overnight at 4°C with a rabbit anti-TNK2 antibody (1:1,000) (Affinity, USA) and a rabbit monoclonal antibody against GAPDH (1:5,000) (Beyotime Biotechnology, Beijing, China). Then, the membranes were washed three times with TBS-T buffer, which was followed by incubation with a goat anti-rabbit secondary antibody (1:5,000, Beyotime Biotechnology) for one h at room temperature. Immunoreactive proteins were detected using an electrochemiluminescence (ECL) Reagent (Affinity, USA) and an automatic chemiluminescence image analysis system (Tanon 5200).

The rats were euthanized using isoflurane anesthesia after the aforementioned tests. The lumbar segments (L3–L5) of whole spinal cords (n = 6/group) were homogenized and centrifuged at 16,000 g and 4°C for 15 min. Equal amounts of protein (50 μg) in supernatants were denatured, resolved by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 5% stacking gel, and 10%–12% separating gel), and then electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Massachusetts, USA). Nonspecific protein binding on the membranes was blocked with 5% nonfat milk in tri-buffered saline and Tween 20 (TBST) for 2 h at room temperature and then incubated with the primary antibodies: rabbit anti-c-Fos, anti-Iba-1, anti-PKCε, anti-STAT3 (1 : 1000; Affinity Biosciences, OH, USA), anti-IL-6 (1 : 1000; Abcam, MA, USA), anti-GFAP, and anti-GAPDH (1 : 1000; Cell Signaling Technology, MA, USA). The blots were washed with TBST and then probed with secondary HRP goat anti-rabbit IgG (1 : 10,000) in 5% nonfat milk in TBST for 1 h at room temperature. The proteins of interest on the blots were visualized using an ECL reagent (Affinity Biosciences) and photographed using an X-ray film. The protein band intensity was quantified using the ImageJ software (National Institutes of Health, MD, USA).