IP was purchased from TargetMol Inc (Boston, MA, USA). Rabbit alkaline phosphatase (ALP), Runx2, GPR30 antibody, bone morphogenetic protein (BMP)-2 antibody were obtained from Abcam Inc (Cambridge, UK). AKT and p-AKT antibody were sourced from Cell Signaling Technologies (Danvers, MA, USA). TRIzol, Reverse Transcriptase, and SYBR® Premix Ex Taq™ were obtained from TakaRa Biotech (Tokyo, Japan). Lipo 2000 and small interfering RNA (siRNA) were supplied by Thermo Fisher Scientific (Waltham, MA, USA). LY294002 was obtained from MedChemExpress (NJ, USA).
Bmp 2 antibody
Manufactured by Abcam
Sourced in United Kingdom
The BMP-2 antibody is a research tool used to detect the presence and levels of BMP-2 (Bone Morphogenetic Protein 2) in biological samples. BMP-2 is a growth factor that plays a crucial role in the regulation of bone and cartilage development.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Lipo2000, by Thermo Fisher Scientific (1 mentions)
Reverse transcriptase, by Takara Bio (1 mentions)
P akt antibody, by Cell Signaling Technology (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Trizol, by Takara Bio (1 mentions)
Ly294002, by MedChemExpress (1 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
Gel pro analyzer 3, by Media Cybernetics (1 mentions)
Ecl system, by Cytiva (1 mentions)
Trans blot sd semi dry electrophoretic transfer cell, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P smad1 5 8, by Cell Signaling Technology (1 mentions)
Aurkb, by Santa Cruz Biotechnology (1 mentions)
β catenin small interfering rna sirna, by Santa Cruz Biotechnology (1 mentions)
Aurka, by Santa Cruz Biotechnology (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
P β catenin, by Cell Signaling Technology (1 mentions)
6 protocols using bmp 2 antibody
1
Osteogenic Differentiation Pathway Analysis
2
Protein Expression Analysis of Developmental Genes
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Western blot analysis was performed to assess the protein expression of Dhcr7, Shh, and Bmp2. The palatal shelf cells were harvested at different time points for protein extraction with the M-PER mammalian protein extraction reagent (Pierce). Equal amounts of total protein were loaded onto a 10% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore) in a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad) at 15 V for 30 min. The membrane was blocked for 2 h at room temperature with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TTBS) and incubated overnight with antibodies (see below). Proteins were then incubated with a peroxidase-conjugated secondary antibody for 1 h and developed with an ECL1 Detection kit (Amersham), according to the manufacturer's instructions. The protein was detected with an ECL system (Amersham) by chemoluminescence and visualized on radiographic film. Protein expression was quantified with Gel-Pro Analyzer 3.1 software (Media Cybernetics). The antibodies used in the present study were Dhcr7 antibody (1 : 2,000, Abcam), Shh antibody (1 : 4,000, Abcam), and Bmp2 antibody (1 : 5,000, Abcam). Monoclonal anti-β-actin antibody (Sigma), diluted 1 : 5000, was used as a loading control.
3
Elucidating BMP-2 Signaling Pathways
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Recombinant human BMP-2 was obtained from Dae Woong Pharmaceuticals (Seoul, South Korea). The BMP-2 antibody was aquired from Abcam (Cambridge, UK) and p-Smad1/5/8, β-catenin, p-β-catenin, ERK1/2, and p-ERK1/2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies for BMPRII, c-Myc, AURKA, AURKB, and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-catenin small interfering RNA (siRNA) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and the aurora kinase A and B inhibitor (AURK inhibitor) was obtained from TOCRIS Bioscience (Bristol, UK).
4
Protein Expression Analysis by Western Blot
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Western blot analysis included protein extraction, electrophoresis, membrane transfer, and immune coloration. Total protein (30 µg) was loaded to SDS-PAGE gel and then transferred to the nitrocellulose membrane. The membrane was incubated at room temperature with TBST containing 50 g/l milk. The membrane was incubated with primary rabbit polyclonal BMP-2 antibody (Abcam, Cambridge, MA, USA; catalog no.: ab14933) overnight at 4°C. The membrane was washed repeatedly with TBST and incubated with corresponding secondary antibody (1:5,000). The antibody was exposed with ECL solution and the gray value was calculated. The assay was performed in triplicate.
5
Esophageal Cancer Cell Signaling Pathway
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Recombinant human BMP-2 was purchased from DaeWoong Pharmaceutical (Seoul, South Korea). Cell cycle related protein antibodies (cyclin D1, CDK4, p21, p53, p-Smad, and CDK6) and Hippo signaling pathway related protein antibodies (Mst1, Mst2, Sav1, LATS1, p-LATS1, Mob1, p-Mob1, YAP, and p-YAP) were obtained from Cell Signaling Technology (Danvers, MA, USA). The BMP-2 antibody was obtained from Abcam (Cambridge, UK) and BMPR II, RASSF1, Akt, p-Akt, TP63, and β-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). RASSF1 small interfering RNA (siRNA), YAP siRNA, or control siRNA were purchased from Santa Cruz biotechnology (Dallas, TX, USA). The human TE-8 and TE-12 esophageal cancer cell lines were obtained from Dr. Izzo (Unversity of Texas M.D. Anderson Cancer Center, Houston, TX, USA). DMEM-F12 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco), 100 mg/ml streptomycin, and 100 IU/ml penicillin (Gibco) was used for cell medium. Cells were maintained under standard conditions at 37 °C in a 5% CO2 humidified atmosphere.
6
Wnt Signaling and Bone Regeneration
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The expression of β-catenin, Wnt4, Wnt5B, PCNA and BMP-2 were detected by Western blot. Brie y, the total protein was extracted from callus tissue samples. The callus tissue was ground in liquid nitrogen, and the RIPA buffer was added to the pyrolysis solution. After centrifugation, the total protein was extracted by using kit (Millipore, Billerica, MA, USA). The supernatant was separated and packed in centrifugal tube and stored at -20 o C. Then, the protein was added to equal weight and separated by twelve alkyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Then the membranes were blocked with 5% defatted milk powder, and incubated with primary antibody including β-catenin antibody (1:300, Abcam, UK), Wnt4 antibody (1:300, Abcam, UK), Wnt5B antibody (1:300, Abcam, UK), PCNA antibody
(1:300, Abcam, UK), BMP-2 antibody (1:300, Abcam, UK) and GAPDH (1:2000, Santa Cruz Biotechnology Inc, USA) at 4°C overnight respectively. After that, the membranes were incubated with horseradish peroxidase-labeled secondary antibody (1:5000, Beijing Zhong Shan Biotechnology Co., Ltd., Beijing, China) at room temperature for 1 h. Proteins were visualized with enhanced chemiluminescence kit and gel imaging system (Invitrogen™ E-Gel™ Imager, ThermoFisher scienti c, US). Results were analyzed by Image Tools (Image J, National Institutes of Health, US).
(1:300, Abcam, UK), BMP-2 antibody (1:300, Abcam, UK) and GAPDH (1:2000, Santa Cruz Biotechnology Inc, USA) at 4°C overnight respectively. After that, the membranes were incubated with horseradish peroxidase-labeled secondary antibody (1:5000, Beijing Zhong Shan Biotechnology Co., Ltd., Beijing, China) at room temperature for 1 h. Proteins were visualized with enhanced chemiluminescence kit and gel imaging system (Invitrogen™ E-Gel™ Imager, ThermoFisher scienti c, US). Results were analyzed by Image Tools (Image J, National Institutes of Health, US).
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