The total protein expression of HIF-1α and the total and nuclear level of protein expression of NF-κB in BMMSCs were measured by Western blotting. The Western blot analysis was performed according to previously established methods [22 (link)]. Briefly, BMMSCs were collected and lysed in M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). All samples were normalized according to the protein concentrations and separated in 10% SDS-PAGE gels and then transferred to nitrocellulose filter membranes (Pall Corporation, Washington, NY) using the wet transfer blotting system (Bio-Rad, Hercules, CA). The following antibodies were used for Western blotting: anti-HIF-1α (Santa Cruz Biotechnology), anti-NF-κB (Abcam), and anti-GAPDH (Abcam). Goat anti-rabbit secondary antibody was obtained from Santa Cruz Biotechnology. The gray levels of blots were analyzed using ImageJ software.
Nitrocellulose filter membrane
Manufactured by Pall Corporation
Sourced in United States
Nitrocellulose filter membrane is a high-performance filtration product designed for laboratory applications. It is a thin, porous membrane made of nitrocellulose material that is commonly used for the filtration and separation of particles, macromolecules, and other analytes from liquid samples. The membrane's core function is to provide efficient and consistent filtration performance to support various analytical and sample preparation procedures in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Wet transfer blotting system, by Bio-Rad (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Anti gapdh, by Abcam (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ab83259, by Abcam (2 mentions)
Ab94744, by Abcam (2 mentions)
Ab23981, by Abcam (2 mentions)
Chemiluminescence detection system, by Merck Group (2 mentions)
No c510003, by Sangon (2 mentions)
Anti hif 1α, by Santa Cruz Biotechnology (1 mentions)
Anti nf κb, by Abcam (1 mentions)
Goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Protein a g bead, by Santa Cruz Biotechnology (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Rabbit anti vinculin, by Cell Signaling Technology (1 mentions)
Mouse anti flag, by Merck Group (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Amersham ecl plus western blotting detection system, by GE Healthcare (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
16 protocols using nitrocellulose filter membrane
1
Measuring HIF-1α and NF-κB in BMMSCs
2
Protein Extraction and Western Blotting
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Western blotting and IP assay were conducted following the protocols [34 (link)]. In brief, the cells or tissues were lysed in RIPA lysis buffer (Beyotime, P0013B) with Protease Inhibitor Cocktail (Roche, 05892970001) and Phosphatase Inhibitor (Roche, 04906845001) for 1 h. After 12,000 rpm centrifugation for 20 min at 4 °C, the supernatant was taken. Then, the protein supernatants were separated using SDS-PAGE and transformed onto nitrocellulose filter membranes (Pall Corporation, 66485). After blocking by 5% BSA for 1 h, the membranes were incubated with primary antibodies at 4 ℃ overnight. The blots were measured by laser scanner (Odyssey, Licor, USA). Cells were lysed in western blot & IP lysis buffer (Beyotime, P0013). The lysates were incubated with protein A/G beads (Santa Cruz, sc-2002) and primary antibody for 12 h at 4 °C. The precipitates were collected by centrifugation at 1500 rpm for 5 min at 4 °C and washed three times with the cold 1×TBS. The protein samples were subjected to western blotting analysis.
3
Western Blot Analysis of FLAG-tagged Proteins
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HEK293T cells were cultured for 36 h after transfection and washed with phosphate-buffered saline (PBS). The cells were lysed in RIPA lysis buffer (Shanghai Wei AO Biological Technology, Shanghai, China) with 1% protease inhibitor cocktail (Bimake, Houston, TX, USA). The protein samples were then mixed with 5× sodium dodecyl sulfate (SDS) loading buffer and heated for 10 min at 100°C. Equal amounts of protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis (EZBiolab, Parsippany, NJ, USA) and transferred to nitrocellulose filter membranes (Pall Corporation, New York, NY, USA). The membranes were blocked with 5% non-fat milk diluted in PBS with 0.1% Tween 20 (PBST) for 1 h. They were then incubated overnight at 4°C with mouse anti-FLAG (1:1000 dilution, Sigma-Aldrich, Castle Hill, NSW, Australia) and rabbit anti-vinculin (1:3000 dilution, Cell Signaling Technology, Danvers, MA, USA) primary antibodies. The membranes were washed three times in PBST and then incubated with goat anti-rabbit/mouse IgG secondary antibodies (1:5000 dilution, Abmart, Shanghai, China) for 1 h at room temperature. Finally, the membranes were imaged on a chemiluminescent imaging system (5200, Tanon, Shanghai, China). Quantitative analysis of the western blotting results was performed with the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Analysis of Protein Expression
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After transfection for 48 h, cells were harvested and lysed with the NP-40 buffer [150 mM NaCl, 1% NP-40, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA] containing 0.15 U/ml aprotinin, 20 mM leupeptin and 1 mM phenylmethylsulfonyl fluoride. Proteins were loaded onto the 15% SDS-PAGE and transferred onto nitrocellulose filter membranes (Pall Life Sciences, Port Washington, NY, USA). The membrane were initially blocked with 5% non-fat milk for 1 h at room temperature (RT) and then incubated with the primary antibody overnight at 4°C. The membranes were then incubated with the secondary antibody for 1 h at RT. The western blot bands were visualized with the Amersham™ ECL Plus Western Blotting Detection System (GE Healthcare, UK). The antibodies used in this study included anti-p27 (cat. no. sc-1641, Santa Cruz Biotechnology, Inc., Dallas, TX, USA; dilution ratio: 1:2,000), anti-GAPDH (cat. no. 3H12, MBL, Japan; dilution ratio: 1:3,000) and anti-Flag (cat. no. ab1257; Abcam, Cambridge, MA, USA; dilution ratio: 1:2,000) which were purchased from the mentioned companies. The intensities of the protein bands were analyzed using the Image J software (version D1.47; National Institutes of Health).
5
Quantitative Protein Analysis in HemECs
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Total protein was extracted from HemECs at 48 h post-transfection using RIPA lysis buffer (Sigma-Aldrich; Merck KGaA). Total protein was quantified using a BCA Protein assay kit (Thermo Fisher Scientific, Inc.) and 20 µg protein/lane was separated using 4–20% precast polyacrylamide gels (Sigma-Aldrich; Merck KGaA). The separated proteins were subsequently transferred onto nitrocellulose filter membranes (Pall Life Sciences) and blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) for 1.5 h. The membranes were then incubated with primary antibodies at 4°C overnight. Following the primary antibody incubation, the membranes were incubated with a secondary antibody for 1.5 h. Protein bands were visualized using ECL western blotting substrate (Thermo Fisher Scientific, Inc.). The protein density was evaluated with ImageJ (Version 1.8.0, National Institutes of Health). The antibodies used for western blotting are listed in Table II .
6
Protein Extraction and Western Blot Analysis
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Cells were washed with phosphate-balanced saline (PBS) and homogenized on ice with lysate buffer (RIPA; C1503, Beijing Applygen Technologies Inc.), 1 mM PMSF, and 2% proteinase inhibitor (539, 134; Calbiochem) . The homogenates were centrifuged at 13,000 rpm for 10 min at 4°C and the supernatants were collected and boiled in SDS-PAGE buffer. Proteins were electrophoresed and transferred to nitrocellulose filter membranes (Pall Life Sciences). The membranes were blocked for 1 h with PBS containing 0.1% Tween-20 (v/v) and 5% fat-free milk (w/v), then incubated with primary antibodies at 4°C overnight in PBST containing 2% bovine serum albumin. After washing with 0.1% Tween-20 containing PBS (PBST), they were then incubated with secondary antibodies at room temperature for 1 h. Blots were scanned with an Odyssey infrared imaging system (LI-COR Biosciences) and quantified with ImageJ (National Institutes of Health, Bethesda, MD). The primary antibodies used were anti-Syb2 (104202, SYSY) and -actin (A5316, Sigma). The secondary antibodies were IRDye 800CW goat anti-rabbit IgG (LIC-926-32211) and IRDye 680CW goat anti-mouse IgG (LIC-926-32220), both from LI-COR Biosciences.
7
Protein Expression Analysis in Bone Cells
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Proteins were extracted using a commercial kit (No.C510003, Sangon Biotech, China) according to the manufacturer's instructions. Primary antibodies rabbit anti-RUNX2 (ab23981, Abcam), rabbit anti-OSX (ab94744, Abcam), rabbit anti-ACAN (ab36861, Abcam), rabbit anti-OCN (23418-1-AP, ProteinTech, Wuhan, China), rabbit anti-ALP (ab83259, Abcam) and rabbit anti-PBX1 (ab97994, Abcam) were used (all at 1:1,000 dilution). The protein samples were separated on 10% SDS-PAGE gels and subsequently transferred to nitrocellulose filter membranes (Pall Corp.,Washington, NY) using the wet transfer blotting system (BioRad, Hercules, CA). After incubation, secondary antibody goat anti-rabbit-HRP (Pierce, USA) was used at 1:2,000 dilution. The proteins were then detected by chemiluminescence detection system (Millipore, USA). Anti-GAPDH was used as an endogenous control.
8
Protein Extraction and Western Blot Analysis of Bone Cell Markers
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Proteins were extracted using a commercial kit (No. C510003, Sangon Biotech, China) according to the manufacturer’s instructions. The following primary antibodies were used (all at a 1:1,000 dilution): rabbit anti-RUNX2 (ab23981, Abcam), rabbit anti-OSX (ab94744, Abcam), rabbit anti-OCN (23418-1-AP, ProteinTech, Wuhan, China), rabbit anti-ALP (ab83259, Abcam), rabbit anti-ACP5 (ab191406, Abcam), rabbit anti-ITBG3BP (ab192324, Abcam), rabbit anti-MMP9 (10375-2-AP, ProteinTech, Wuhan, China), rabbit anti-CTSK (ab19027, Abcam), rabbit anti-GAPDH (10494-1-AP, ProteinTech, Wuhan, China), rabbit anti-FOXO3 (10849-1-AP, ProteinTech, Wuhan, China) and rabbit anti-TAK1 (ab109526, Abcam). The protein samples were separated by 10% SDS–PAGE and subsequently transferred to nitrocellulose filter membranes (Pall Corp., Washington, NY) using a wet transfer blotting system (Bio-Rad, Hercules, CA). After incubation, a goat anti-rabbit-HRP secondary antibody (Pierce, USA) was applied at a 1:2,000 dilution. The proteins were then detected by a chemiluminescence detection system (Millipore, USA). Anti-GAPDH was used as an endogenous control. The uncropped scans of all blots were provided in the Source Data file.
9
Western Blot Analysis of CCR1, CCR2, and p-p65
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The Western blot analysis was performed according to previously established methods [26 (link)]. Briefly, cells were collected and lysed in M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). All samples were normalized according to the protein concentrations and separated in 10% SDS-PAGE gels and then transferred to nitrocellulose filter membranes (Pall Corp., Washington, NY) using the wet transfer blotting system (Bio-Rad, Hercules, CA). The following antibodies were used for Western blotting: anti-CCR1, anti-CCR2 (Santa Cruz Biotechnology), anti-p-p65 (Abcam), and anti-GAPDH (Abcam) as an endogenous control. Goat anti-mouse CCR2 antibody was obtained from Santa Cruz Biotechnology. The gray levels of blots were analyzed using Image J software.
10
Protein Expression Analysis of Choke Vessels
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The tissues of “choke vessels 1” were lysed for extraction of total cellular protein using tissue extraction kit (Biovision Corporation, California, USA). The proteins were processed using sodium dodecyl sulphate–polyacrylamide gel (Sigma-Aldrich, Missouri, USA) electrophoresis and transferred on to a nitrocellulose filter membrane (Pall corporation, New York, USA). The membrane was blocked with 5% skim milk (Becton, Dickinson and Company, New Jersey, USA) for 2 h and incubated sequentially with monoclonal rabbit anti-rat primary antibodies against HIF-1α (1:1000, Abcam, USA), VEGF (1:1000, Abcam, USA) and β-actin (1:1000, Santa Cruz Biotechnology), and then probed with goat anti-rabbit secondary antibody. Antibody-binding bands were detected using enhanced chemiluminescence plus reagent (Advansta Corporation, California, USA). Intensities of target bands relative to β-actin were analyzed using Image J (Image Processing and Analysis in Java, National Institutes of Health) to calculate the gray values. The experiment was independently repeated three times.
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