Nitrocellulose transfer membrane

Human umbilical arteries were lysed in 1 × RIPA buffer with 1μg/mL leupeptin and 1μg/mL phenylmethylsulfonyl fluoride. Aliquots containing 30 μg of protein samples were separated by 12% SDS-PAGE and transferred electrophoretically to a nitrocellulose transfer membrane (Bio-Rad, Hercules, CA, USA). After blocked with 5% BSA in TBS containing 0.01% Tween 20 (TBST) for 1 h at room temperature, the membrane was incubated overnight at 4^°C with primary antibodies against FGA (1:1,000, Abcam, Cambridge, MA, USA), ACTA2 (1:1,000, Abcam, Cambridge, MA, USA), IDH3A (1:1,000, Abcam, Cambridge, MA, USA), GAPDH (1:1,000, Abcam, Cambridge, MA, USA). Subsequently, membranes were washed 10 min for three times with TBST, then each membrane was incubated for 1 h at room temperature with the appropriate secondary antibody (anti-rabbit IgG, anti-mouse IgG; 1:5,000; Abcam). The protein intensities were determined and analyzed using Odyssey^® Imager (LI-COR Biosciences, Lincoln, NE, USA).

Western blotting was performed as we previously described (Pan et al., 2018 (link)), four exosomes of human serum were prepared from control groups and four samples from PCOS groups, separately. Samples were separated in a 10% SDS gel. The separated samples were transferred to a nitrocellulose transfer membrane (Bio-Rad, Hercules, CA, USA). After incubating for 1 h with blocking buffer, the membrane was incubated overnight at 4 °C with primary antibodies against Hsp70 (Beyotime AF1156, Shanghai, China, 1:1,000), CD9 (Beyotime AF1192, Shanghai, China, 1:1,000) , beta Tubulin (Beyotime AF1216 , Shanghai, China, 1:1,000) and Calnexin (Beyotime AC018, Shanghai, China, 1:1,000) at 4 °C overnight. After three washes with −TBST,pH 7.4, each membrane was incubated with the appropriate secondary antibody (1: 1000) at room temperature for 1 h. After additional three washes, protein intensities were visualized with the enhanced ECL detection system (Sage Creation, Beijing, China). Each experiment was repeated three times.

For western blot analysis, proteins obtain from the IK and primary cultured human endometrium cells were electrophoresed at 20 μg/lane and separated on a 15% SDS gel. After running on a gel, the proteins were transferred to a nitrocellulose transfer membrane (Bio-Rad, Hercules, CA, USA). To protect from non-specific binding the membrane was incubated for 1 h with 5% skimmed milk (Difco) in Tween20 1x TBS (TBST). Next, the membrane was incubated overnight at 4°C with primary antibodies against CDKN2a (Abcam ab108349, Cambridge, UK, 1:1000), EPCR (Abcam, ab174234, Cambridge, UK, 1:2000), ARVCF (Santa Cruz Biotechnology, INC sc-23874, California, USA; 1:1000) and GAPDH (CW bio CW0266A, Beijing, China, 1:1000) at 4°C overnight. After three washes with 1x TBST (pH 7.4), each membrane was incubated with the appropriate secondary antibody (1: 10000) at room temperature for 1 h. After additional three washes, protein intensities were determined and analyzed using Odyssey^® Imager (LI-COR, Lincoln, NE, USA).

The cell extracts were prepared by lysing unsorted epithelial cells with RIPA buffer containing 150 mM NaCl, 50 mM Tris–HCl (pH = 8), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitors and phosphatase inhibitors (Sigma). The cell extract (20 µg) was run on an 8–10% SDS-PAGE gel and transferred to a nitrocellulose transfer membrane (Bio-Rad). After incubating for 1 h with blocking buffer, the membrane was incubated overnight at 4°C with mouse monoclonal anti-complement C₃ (1:500 Santa Cruz Biotechnology sc28294), mouse monoclonal anti-plasminogen (1:500 Santa Cruz Biotechnology sc376324), rabbit polyclonal anti-kininogen 1 (1:1000 Abcam ab97761) and mouse polyclonal anti-GAPDH antibody (1:5000 Novus Biologicals, Littleton, CO, USA). After three washes with 1× TBST, pH 7.4, the samples were then incubated with fluorescence-labeled anti-mouse IgG or anti-rabbit IgG antibody (Daylight 680 or 800, KPL; 1:5000) for 1 h at room temperature and analyzed with an Odyssey Imager (Li-Cor; Lincoln, NE, USA).

Protein lysates were extracted from HCT-116 cells using RIPA buffer with a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). After measuring protein concentration with a Pierce^® BCA Protein Assay Kit (Thermo Fisher Scientific Inc, Rockford, IL, USA), 20 µg protein were added to 6× Laemmli buffer (Bio-Rad, Hercules, CA, USA), denatured and loaded to a 7.5% Mini-Protean TGX Precast Gel (Bio-Rad, Hercules, CA, USA). After electrophoresis at 100V gel was blotted to a nitrocellulose transfer membrane (Bio-Rad, Hercules, CA, USA), blocked and incubated with primary (see immunohistochemistry protocol; dilution 1:1000, and actin antibody, Cell Signaling Technology, Inc., Danvers, MA, USA) and secondary antibodies (goat anti-rabbit, ab6721, Abcam, Cambridge, UK, dilution 1:1000). Chemiluminescence and detection were performed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) and a CCD camera. Data analysis was performed using ImageJ software 1.53 (NIH, Bethesda, MD, USA) and standardized to actin.

Mouse liver homogenates were prepared using a RIPA buffer containing protease and tyrosine phosphatase inhibitor cocktail (Sigma). The protein concentration of the lysates was determined using the Bicinchoninic (BCA) Protein Assay Kit (Thermo Scientific, IL, USA), according to the manufacturer’s instructions. The isolated soluble proteins (20 mg) were separated on 8–15% SDS-polyacrylamide gels. The separated proteins were then electroblotted onto nitrocellulose transfer membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated with 5% skim milk for 1 h and then probed with the following 1:1,000-diluted antibodies: anti-pAMPKa, anti-AMPKa, anti-pACC, anti-ACC, anti-CYP2E1, anti-CPT1a (Cell Signaling Technology), and anti-β-actin (Santa Cruz Biotechnology) for 18 h at 4°C. After 3 washes for 10 min each, polyclonal anti-rabbit or mouse HRP-conjugated secondary antibodies (Santa Cruz Biotechnology), which were linked to HRP-bound protein complexes and developed with a Pierce ECL Western Blot substrate (Thermo Fisher Scientific), were added to the mixture. Band densitometry was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA) and expressed as fold change relative to β-actin.

Total protein concentrations in tissue lysates were measured by Lowry assay. Samples of the protein (150 μg/ml) were then separated by using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose transfer membranes (Bio-Rad Laboratories, Inc. USA). The membranes were then probed by using antibodies against ECE-1 (Cat. 6855b, ABGENT, San Diego, CA, USA), dilution: 1 : 1000 in 0.1% BSA in PBS and VEGF (Cat. 115544, Biorbyt, Germany; 1 : 500 in milk 4%). Immune complexes were detected with goat anti-rabbit IgG HRP (Cat. HAF008, R&D Systems, USA; 1 : 1000 BSA 4%). The blots were then visualized by using chemiluminescence (ChemiDoc™ XRS + System, Bio-Rad Laboratories, Inc. USA), and the signal intensity was quantified by densitometry using Image Lab™ Software (Bio-Rad Laboratories, Inc. USA).