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21 protocols using WESTSAVE up

Western blotting analyses were conducted to detect proMMP-2 and -9 in cellular lysate using anti-MMP-2 (ALX-210-753, Enzo Life Sciences, Farmingdale, NY, USA) and anti-MMP-9 (3852S, Cell Signaling Technology, Danvers, MA, USA) antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used as an internal loading standard, was detected using anti-GAPDH antibody (LF-PA0212, AbFrontier, Seoul, Korea). Cellular lysates were separated on 10% (w/v) SDS-PAGE and electrotransferred to PVDF membrane. After the blotted membrane was probed with primary antibodies overnight at 4 °C, it was incubated with secondary antibody (goat anti-rabbit IgG-pAb-HRP-conjugate; ADI-SAB-300, Enzo Life Sciences, Farmingdale, NY, USA) for 1 h at room temperature, and developed using an enhanced West-save up™ (AbFrontier, Seoul, Korea).
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Equal quantities of nuclear and cytosolic extracts (20 μg) were electroblotted onto a nitrocellulose membrane, following separation using 8–12% sodium dodecylsulfate-polyacrylamide gel electrophoresis. The blot was probed using primary antibodies against HO-1, Nrf2, Lamin B, p-p65, p65, p-eNOS, eNOS, p-IRS-1, IRS-1, and β-actin. HRP-conjugated anti-IgG antibodies were used as the secondary antibodies to detect the previously mentioned protein bands by enhanced chemiluminescence (WESTSAVE-Up; AbFrontier, Seoul, Korea).
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Protein lysates were prepared according to the protein extraction protocol (Cell Signaling Technology, Beverly, MA, USA) with 1 mM phenylmethylsulfonyl fluoride and 1×protease inhibitors (Sigma-Aldrich) and quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Thereafter, 30–50 μg of protein lysate was separated by electrophoresis on a 10% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride membranes, which were then blocked with 5% nonfat skim milk and probed with the following primary antibodies at the indicated dilutions: cAMP-responsive element-binding protein (CREB), 1:1000 (#9197); p-CREB, 1:1000 (#9198, all from Cell Signaling Technology, Danvers, MA, USA); and β-actin conjugated horseradish peroxidase (HRP), 1:10000 (47778; Santa Cruz, Paso Robles, CA, USA). The secondary antibody used was donkey anti-rabbit immunoglobulin-horseradish peroxidase antibody (1:5000; Santa Cruz). After incubation with the primary and secondary antibodies, the blots were visualized using a western blotting substrate (WESTSAVEup, AbFrontier, Seoul, South Korea) and exposed to X-ray film (Agfa Healthcare, Mortsel, Belgium). Proteins of interest were quantified using ImageJ v1.51.
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After the NMDA treatment, the SH-SY5Y cells were washed with PBS for three times followed by protein extraction with lysis buffer. Equivalent amounts of samples (50 µg/well) were applied onto a 10% SDS-Polyacrilamide gel. After SDS-PAGE, the proteins were electroblotted to PVDF membranes (Bio-Rad) and blocked with 5% non-fat skim milk overnight. To analyze the expressions of different target proteins, the membranes were subsequently incubated in the primary antibodies supplemented with 2% Bovine Serum Albumin (Sigma). After washing with Tris-Buffered Saline and Tween 20 (TBST), the respective horseradish peroxidase (HRP)-conjugated secondary antibodies supplemented with 2% non-fat skim milk were applied onto the membrane at room temperature. The bands on the membranes were then visualized by Westsave Up (Abfrontier, South Korea). The immunoblotting of myosine IIb showed a form of multiple bands because of the existence of myosin IIb polymer. The quantification of myosine IIb was measured from the major band (200 kDa).
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Cell disruption buffer (Thermo Fisher Scientific) with proteinase inhibitor (Merck, Darmstadt, Germany) and phosphatase inhibitor (Merck) was used to lyse the uterine horns. After denatured at 95°C for 10 min, equal amounts of proteins were subjected to 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk for 1 h at room temperature, followed by an overnight incubation with primary antibodies at appropriate concentrations (supplementary table S3) at 4oC. The membranes were incubated with corresponding HRP-conjugated secondary antibodies (listed in supplementary table S3) at room temperature for 1 h. Protein bands were then visualized using ECL detection reagents (Westsave UP™; AbFrontier, Seoul, Korea) and immediately exposed to x-ray film (Shenzhen Fumingwei Industrial Co., LTD, Shenzhen, China). Quantification of protein bands was achieved using ImageJ software and the expression of target proteins were calculated relative to internal control β-actin.
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Western blotting analyses were performed to detect iNOS and filaggrin in cellular lysates using anti-iNOS (610332, BD Transduction Laboratories, KY, USA) and anti-filaggrin (SC-30229, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies, respectively. GAPDH, used as an internal loading standard, was detected using anti-GAPDH antibody (LF-PA0212, AbFrontier, Seoul, Korea). After the blotted membrane was incubated with primary antibodies overnight at 4°C, it was reacted with secondary antibody (goat anti-rabbit IgG-pAb-HRP-conjugate; ADI-SAB-300, Enzo Life Sciences, Farmingdale, NY, USA) for 1 h at room temperature and developed using an enhanced West-save up ™ (AbFrontier, Seoul, Korea).
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To detect MMP-2 and Nrf2 proteins in the cellular lysate, western blotting analysis was performed using anti-MMP-2 (ALX-210-753, Enzo Life Sciences, Farmingdale, NY, USA) and ant-Nrf2 (ab31163, Abcam, Cambridge, MA, USA) antibodies as the primary antibodies. The cellular lysates were run on a 10% (w/v) SDS-PAGE and electrotransferred to PVDF membranes. The membranes were blocked with blocking buffer (2% BSA in 1X TBS-Tween 20), probed with primary antibody overnight at 4℃, incubated with secondary antibody (goat anti-rabbit IgG-pAb-HRP-conjugate; ADI-SAB-300, Enzo Life Sciences, Farmingdale, NY, USA) for 1 h at room temperature, and developed using an enhanced West-save up™ (AbFrontier, Seoul, Korea).
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Histone proteins were extracted from the leaf tissues of 2-week-old plants as previously described (Tariq et al., 2003 (link)). After being washed in acetone and dried, the proteins were resuspended in Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue, and 10% β-mercaptoethanol), then separated on a 15% SDS-PAGE and transferred to an Immobilon-P PVDF transfer membrane (Millipore). The membrane was blocked with 2% bovine serum albumin in phosphate-buffered saline (pH 7.5), and incubated overnight with primary antibodies against dimethyl-histone H3K9, trimethyl-histone H3K4, trimethyl-histone H3K27, and histone H3 (Millipore; catalog nos. 07–441, 07–473, 07–449, and 06–775, respectively) at a 1:5,000 dilution at room temperature. After three washes (30 min each), the secondary antibody [goat anti-rabbit IgG (Southern Biotech)] at a 1:10,000 dilution was used. For immunoblotting detection, we used an enhanced chemiluminescence detection system (WESTSAVE-Up; AbFrontier). The intensities of each protein were calculated using Image J (version 1.51s) software according to the instructions3. The levels of each protein are shown relative to those of WT, which is set as 1, and are indicated on the bottom of each protein blot.
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Proteins from in vitro differentiated FPC and non-FPC were extracted with cell lysis buffer (Ambion, Grand Island, NY, USA). The protein samples (5 μg) were mixed with 5X SDS loading buffer (60 mM Tris–HCl pH 6.8, 2% SDS, 0.1% bromophenol blue, 25% glycerol and 14.4 mM β-mercaptoethanol) and denatured at 95°C for 10 minutes. Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk (Nestle, Vevey, Switzerland) in PBS containing 0.1% Tween-20 (PBST) for 30 minutes and incubated with primary antibodies at appropriate concentrations (Additional file 5: Table S3) overnight at 4°C. The membranes were stained with appropriate horseradish peroxidase-conjugated secondary antibodies (Additional file 5: Table S3) for one hour at room temperature. The protein bands were visualized by enhanced luminal-based chemiluminescence (Westsave UP™; AbFrontier, Seoul, Korea). Endometrial stromal cells cultured in serum containing medium were used as the control. Non-immune immunoglobulin of the same isotype as the primary antibody was used as negative control. The scanned Western blot bands were quantified densitometrically and the values were normalized to the amount of β actin using Image J software (US National Institutes of Health, USA).
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Cells were lysed with the lysis buffer containing the premade 2X lysis buffer (150 mM NaCl, 10 mM Tris-HCI (pH 7.4), 1 mM EDTA, and 1 mM EGTA), 1% Triton X-100, 1 mM PMSF, 0.1% DCA, and 1X EDTA-free protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The concentration of protein was determined using BCA Protein Assay Kit (Thermo Fisher Scientific). 10–20 µg of proteins were loaded onto 6–15% SDS-PAGE gels for separation and transferred to nitrocellulose membranes (GE Healthcare Life Sciences) for detection by immunoblotting. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20, incubated with primary antibodies overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibodies for 2 h at room temperature. Signals were visualized with the enhanced chemiluminescence detection kits, WESTSAVE up (AbFrontier) and ECL Select Western Blotting Detection Reagent (GE Healthcare Life Sciences).
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