Cells were washed with ice-cold Dulbecco's PBS and total protein lysates were extracted with cell lysis buffer RIPA (Biocolors, R0095) containing 1% PMSF (Meilunbio, MA0001). For western blot analysis, 20 ug of proteins was resolved on 10% SDS–PAGE gels (Bio-Rad, Richmond, CA) and transferred to polyvinylidene difluoride membranes (Merch, ISEQ00010).The membranes were blocked with TBS containing 5% (w/v) non-fat dry milk and 0.1% Tween-20 for 1 h, and then incubated at 4°C overnight with the appropriate antibodies, including CHRDL1 (Abcam, ab103369), BMPRII (CST, 6979), RUNX2 (Santa Cruz, 10758), p-SMAD1/5/9 (CST, 13820), SMAD1/5/9 (Santa Cruz, 6031), and GAPDH (CST, 5174). Blots were developed with horseradish peroxidase-labeled secondary antibody and visualized using the enhanced chemiluminescence detection system (Millipore, Billerica, MA) according to the manufactures' instructions.
Runx2
Manufactured by Santa Cruz Biotechnology
Sourced in United States
RUNX2 is a protein-coding gene that plays a key role in the regulation of osteoblast differentiation and skeletal morphogenesis. It is a transcription factor that acts as a master regulator of bone formation.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (14 mentions)
Osterix, by Santa Cruz Biotechnology (8 mentions)
Bmp 2, by Santa Cruz Biotechnology (6 mentions)
β catenin, by Santa Cruz Biotechnology (6 mentions)
Col1a1, by Santa Cruz Biotechnology (5 mentions)
Bmp2 4, by Santa Cruz Biotechnology (4 mentions)
Pparγ, by Santa Cruz Biotechnology (4 mentions)
Osteocalcin, by Santa Cruz Biotechnology (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
β actin, by Abcam (3 mentions)
Skim milk, by BD (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Topro, by Thermo Fisher Scientific (3 mentions)
Alliance 2, by Uvitec (3 mentions)
Collagen 1, by Abcam (2 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (2 mentions)
Ecl system, by Thermo Fisher Scientific (2 mentions)
Peroxidase substrate for chemiluminescent detection, by Thermo Fisher Scientific (2 mentions)
Transfer apparatus, by Bio-Rad (2 mentions)
Goat anti rat brdu, by Santa Cruz Biotechnology (2 mentions)
76 protocols using runx2
1
Western Blot Analysis of Osteogenic Regulators
2
Western Blot Analysis of Osteogenic Regulators
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To detect Runx2, HDAC5, and Hoxa2 protein expression, Western blot analysis was performed as previously described [16 (link), 17 (link)]. Briefly, equal amounts of protein (40 μg/lane) were subjected to SDS gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, GE Healthcare Biosciences, Pittsburgh, PA, USA). The membranes were incubated with primary antibodies against Runx2 (Santa Cruz), HDAC5 (Santa Cruz), Hoxa2 (Santa Cruz), and β-actin (Abcam) overnight at 4°C and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) for 1 h at room temperature. The blots then were visualized with the chemiluminescent detection method using the SuperSignal West Pico Substrate (Pierce Biotechnology).
3
Aortic Valve Tissue Analysis
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After euthanasia, thoracotomy was performed to remove the heart and proximal aorta, which were frozen in optimal cutting temperature compound (10.24% polyvinyl alcohol, 4.26% polyethylene glycol, and 85.5% nonreactive ingredients). The apical two thirds of the heart was removed, and remaining tissue was secured in a cryostat. Sections, 10 μm thick, were obtained from proximal, mid, and distal aortic valve from each mouse. Slides were stained with Alizarin Red or Picrosirius Red to quantitate the amount of calcium or collagen, respectively, as described previously.12 Movat's Pentachrome stain was used to visualize proteoglycans and collagen in the aortic valve, as described previously.13 Sources of primary antibodies for immunostaining were as follows: collagen‐1 (Abcam, Cambridge, MA), α‐smooth muscle actin (α‐SMA; Abcam), Runx2 (Santa Cruz Biotechnology, Dallas, TX), and CD31 (BD Biosciences, San Jose, CA).
To detect phosphorylated EGFR in aortic valve, hearts from control mice and Velvet mice, aged 2.5 to 4 months, were incubated in DMEM in the presence (N=5 control, and N=6 Velvet) or absence (N=5 control, and N=5 Velvet) of recombinant EGF (40 ng/mL; R&D Systems, Minneapolis, MN), for 12 minutes, then frozen in optimal cutting temperature compound, cut in 10‐μm sections, and stained with anti–phosphorylated EGFR antibody (Cell Signaling, Danvers, MA).
To detect phosphorylated EGFR in aortic valve, hearts from control mice and Velvet mice, aged 2.5 to 4 months, were incubated in DMEM in the presence (N=5 control, and N=6 Velvet) or absence (N=5 control, and N=5 Velvet) of recombinant EGF (40 ng/mL; R&D Systems, Minneapolis, MN), for 12 minutes, then frozen in optimal cutting temperature compound, cut in 10‐μm sections, and stained with anti–phosphorylated EGFR antibody (Cell Signaling, Danvers, MA).
4
Osteogenic Differentiation Protein Analysis
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After 7 days in differentiation medium with osteogenic induction, two cell groups (miR-93-5p NC group and miR-93-5p mimic group) were seeded on 60 mm plastic dishes (WHB) and cultured for 7 days in osteogenic differentiation medium. Total protein was isolated using RIPA buffer. Proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane for 1.5 h at 4°C. Membranes were blocked with 5% milk in TBST for 2 h at room temperature and incubated with primary antibodies against BMP-2 (1:200, Santa Cruz, USA), OPN (1:200, Santa Cruz, USA), Runx-2 (1:200; Santa Cruz, USA), ALP (1:100, Boster, China), Osterix (1:200, Santa Cruz, USA), or GAPDH (1:3000, Tianjin Sungene, China) at 4°C overnight. Membranes were incubated with HRP-conjugated secondary antibody (1:2000, Boster, China) for 1 h at room temperature, followed by scanning with X-ray film. The integrated intensity for each detected band was then determined with ImageJ, v.1.46.
5
Gene Expression and Chromatin Immunoprecipitation
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Conditions for real time RT-PCR, and immunofluorescence (Hu et al. 2012 (link)). The list of gene-specific primers is provided in Supplemental Table 1 . We used the following antibodies: Collagen Type I (GeneTex, 1:1000), Wnt5a (R&D systems, 1:200), Runx2 (SantaCruz, 1:200). Conditions for chromatin immunoprecipitation (ChIP) were as previously described (Walker et al. 2019 (link)).
6
Western Blot Analysis of Osteogenic Markers
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Proteins (30 μg) derived from all experimental groups were processed as previously described.35 (link) All antibodies used for western blot procedure were purchased to Santa Cruz Biotechnology. After protein separation, saturated sheets were incubated overnight at 4°C with COL1A1 (1:200, Santa Cruz Biotechnology), RUNX2 (1:1000, Santa Cruz Biotechnology), BMP2/4 (1:750, Santa Cruz Biotechnology), OPN (1:750, Santa Cruz Biotechnology) and β-Actin (1:1000, Santa Cruz Biotechnology).36 Then samples were washed and incubated in secondary antibody diluted 1:1000 in 1x TBS, 5% milk, 0.05% Tween-20. Protein specific bands were visualized by means the electrochemiluminescence method.37 (link)
7
Mouse Limb Immunofluorescent Analysis
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Mouse limb sections at P1 were fixed in 10% formalin, stored in 70% ethanol, and subjected to dehydration, paraffin embedding, and sectioning without decalcification for immunofluorescent analysis. A concentration of 1:100 was used for primary anti-Sox9 (sc-166505, Santa Cruz, CA, USA) and Runx2 (sc-390351, Santa Cruz). Non-immune mouse IgG was used as a negative control. Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preabsorbed (1:500, ab150117, Abcam) was used as a secondary antibody, and nuclei were stained with 49, 6-diamidino-2-phenylindole (DAPI) for detection. Image observation and analysis were collected under Nikon Eclipse 80i microscope (Nikon Instruments Inc., Melville, NY, USA).
8
BMSC Lineage Regulation by PPAR-γ and Runx2
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The induced BMSCs were rinsed twice with PBS and scraped in the previously described lysis buffer (Sigma‐Aldrich). After centrifugation at 25,000 × g for 20 min., the protein content of the obtained extracts was determined with a protein assay kit (Bio‐Rad, Canada). For Western blot analysis, the proteins were immunolabelled with monoclonal antibodies directed against peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) and runt‐related transcription factor 2 (Runx2; Santa Cruz Biotechnology). The method was similar to that used for the determination of P‐gp expression. Peroxisome proliferator‐activated receptor‐γ and Runx2 expression was measured by densitometry and normalized to that of GAPDH.
9
Comprehensive Western Blot Protocol
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Western blots were conducted as previously described [18 (link)]. Total protein was extracted using the radioimmunoprecipitation assay buffer. The extract was centrifuged (14,000 rpm) at 4 °C for 30 min. The supernatant was collected, and protein levels were quantified using the Bradford assay. Proteins were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane for 2 h. Subsequently, the membrane was blocked with 3% BSA in phosphate-buffered saline containing 0.1% Tween 20 (PBST) for 2 h at room temperature and incubated with primary antibodies at 4 °C overnight. After washing thrice with PBST, the membrane was incubated with secondary antibodies at 37 °C for 4 h. Antibodies against β-catenin, p-Smad1/5, and p-Smad2/3 and secondary anti-rabbit antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and BMP2/4, TGFβ, RUNX2, OSX, OPN, Col1a1, β-actin and secondary anti-mouse antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Bands were detected using a Pierce® ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). The resulting bands were normalized using β-actin as a loading control.
10
Osteogenic and angiogenic potential of stem cells
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Cells were cultured for a period of 7, 14, or 21 days in 3 different conditioned media: (i) medium C + melatonin (medium M), (ii) medium C + HA + BU + RA mixture (medium H), and (iii) medium C + melatonin + HA + BU + RA mixture (medium MH). Control cells were maintained undifferentiated in the presence of medium C. After 7, 14, or 21 days, the cells were treated with trypsin, and the resulting suspension was cultured at low density to allow visualization of individual cells. Cells were fixed with 100% methanol at −20°C for 30 min and then at −80° for 30 min. After permeabilization by PBS containing 0.25% Triton X-100, cells were washed in PBS three times for 5 min and incubated with 1% BSA in PBS-T (PBS + 0.1% Tween 20) for 30 min and then exposed overnight at 4°C to the primary anti-mouse monoclonal antibodies BSPII (Santa Cruz Biotechnology), osteocalcin (Santa Cruz Biotechnology), and Runx2 (Santa Cruz Biotechnology) or to the rabbit polyclonal antibodies against VEGF A (Thermo Scientific). Cells were then washed in PBS three times for 5 min and stained at 37°C for one hour in the dark with the fluorescence-conjugated goat IgG secondary antibody. All microscopy analyses were performed with a confocal microscope (TCS SP5, Leica, Nussloch, Germany). DNA was visualized with 1 μg/mL 4,6-diamidino-2-phenylindole (DAPI).
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