Immunoblotting was performed as previously described (12 (link)). Equal amounts of tissue lysates were used for immunoblotting. Blots were incubated with specific antibodies against SMAD1, GSK3α, and GSK3β (all from Cell Signaling Technology, 9743, 433T, and 93115); β-catenin and Cbfa1 (all from R&D Systems, AF1329 and MAB2006); osterix (Santa Cruz Biotechnology, sc-22536); Flk1 and VE-cadherin (all from BD Biosciences, 55307 and 562242); and vWF (Dako, A0082). β-Actin (MilliporeSigma, A2228) was used as a loading control. Immunofluorescence was performed as previously described in detail (12 (link)). We used specific antibodies against CD31 (BD Biosciences, 553370), osterix (Santa Cruz Biotechnology, sc-22536), and vWF (Dako, A0082). The nuclei were stained with DAPI (MilliporeSigma, D9564).
Osterix
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
Osterix is a transcription factor that plays a crucial role in the differentiation and maturation of osteoblasts, the cells responsible for bone formation. It is an essential regulator of bone development and homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Runx2, by Santa Cruz Biotechnology (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
β actin, by Merck Group (4 mentions)
Bmp 2, by Santa Cruz Biotechnology (3 mentions)
β catenin, by R&D Systems (3 mentions)
β catenin, by Santa Cruz Biotechnology (3 mentions)
Runx2, by Cell Signaling Technology (2 mentions)
β actin, by Bioworld Technology (2 mentions)
Pecam 1, by BD (2 mentions)
Flk1 and ve cadherin, by BD (2 mentions)
Smad1, by Cell Signaling Technology (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Boster Bio (1 mentions)
Ab11042, by Abcam (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Fab3628g, by R&D Systems (1 mentions)
Alphasma cy3, by Merck Group (1 mentions)
Ter 119, by R&D Systems (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
27 protocols using osterix
1
Immunoblotting and Immunofluorescence of Osteoblast Markers
2
Osteogenic Differentiation Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
3
Bone Tissue Imaging and Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly dissected tibiae were collected from wild type, mutant mice and their control littermates. Bone were processed and imaged as reported earlier8 (link)9 (link). The following primary antibodies were used: Emcn (sc-65495, Santa Cruz, diluted 1:100), Pecam1 conjugated to Alexa Fluor488 (FAB3628G, R&D Systems, 1:100), Pecam1 (553370, BD Pharmingen, 1:100), Osterix (sc-22536-R, Santa Cruz, 1:200), alphaSMA-Cy3 (C6198, Sigma, 1:100), Calcitonin receptor (ab11042, Abcam, 1:75), Dll4 (AF1389, R&D Systems, 1:100), Biotin-conjugated CD45 (553077, Becton Dickinson, 1:100) and Ter-119 (MAB1125, R&D Systems, 1:100). After sections had been washed with PBS for three times, the following secondary antibodies were used: Anti-rat Alexa Fluor 594 (Invitrogen; A21209, 1:400), anti-rabbit Alexa Fluor 546 (Invitrogen; A11035, 1:400), anti-goat Alexa Fluor 488 (Invitrogen; A11055, 1:400) and streptavidin Alexa Fluor 488 (Invitrogen; S11223, 1:400). Immunofluorescent stainings were analysed at high resolution with a Zeiss laser scanning confocal microscope, LSM780.
Proliferating cells were labelled by injecting mice intraperitoneally with EdU (Invitrogen) 3 h before euthanasia. Mice were killed; bones were collected and processed following the protocol mentioned before. For immunostaining EdU using Click-iT chemistry, manufacturer's protocols were followed.
Proliferating cells were labelled by injecting mice intraperitoneally with EdU (Invitrogen) 3 h before euthanasia. Mice were killed; bones were collected and processed following the protocol mentioned before. For immunostaining EdU using Click-iT chemistry, manufacturer's protocols were followed.
4
Bone Differentiation Pathway Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Culture materials and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). MAPK inhibitors including PD98059 (inhibitor for ERK), SP600125 (inhibitor for JNK), SB203580 (inhibitor for p38), and LDN193189 (inhibitor for ALK3) were purchased from Calbiochem (La Jolla, CA, USA). Antibodies against p38, phospho-p38, SMAD1/5/8, phospho-SMAD1/5/8, osterix, and OPN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody against Runx2 was purchased from Cell Signaling Technology (Beverly, MA, USA). The catalog numbers and dilutions for antibodies used in Western blot analysis were provided in Table S1 . Neutralizing mABs against BMP-2, BMP-6 were purchased from R&D Systems (Minneapolis, MN, USA). Runx2, SMAD1 siRNA vectors, and a control siRNA construct (containing random DNA sequences) were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals of reagent grade were obtained from Sigma (St. Louis, MO, USA).
5
Western Blot Analysis of ADSC Osteogenic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the indicated treatment, ADSCs were washed twice with prechilled PBS buffer. The harvested ADSCs were lysed on ice for 30 min in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 100 mM PMSF (Beyotime Biotechnology) to extract total proteins. The protein concentrations were measured by the BCA protein assay kit (Solarbio, Beijing, China). Then, equal amounts of protein extracts (25 μg) were subjected to 10% SDS-PAGE as described. Next, the proteins were transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA).
After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with specific primary antibodies against RUNX2, ALP, and Osterix (Santa Cruz, CA, USA) overnight at 4 °C. The membranes were then washed three times with TBS-Tween 20 and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized with ECL plus (GE Life Sciences) according to the manufacturer’s instructions. The protein band densities were analyzed using ImageJ software (version 18.0, National Institutes of Health, Bethesda, MD, USA).
After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with specific primary antibodies against RUNX2, ALP, and Osterix (Santa Cruz, CA, USA) overnight at 4 °C. The membranes were then washed three times with TBS-Tween 20 and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized with ECL plus (GE Life Sciences) according to the manufacturer’s instructions. The protein band densities were analyzed using ImageJ software (version 18.0, National Institutes of Health, Bethesda, MD, USA).
6
Osteogenesis Protein Abundance Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein abundance of osteogenesis‐related markers was also tested by western blotting. The femoral head was crushed in liquid nitrogen using a mortar and dissolved in immunoprecipitation assay buffer, and protein was extracted. The protein concentration was measured using the bicinchoninic acid quantitative assay. Protein samples (20 μg) were separated using 12% SDS/PAGE and transferred onto a polyvinylidene fluoride membrane. Blocking was performed using 5% skim milk powder for 1 h at room temperature. The membrane was incubated with primary antibodies against glyceraldehyde‐3 phosphate dehydrogenase (dilution 1 : 1400, KM9002; Tianjin Sungene Biotech Co., Ltd., Tianjin, China), BMP‐2 (dilution 1 : 1000, sc‐137087; Santa Cruz Biotechnology), RUNX‐2 (dilution 1 : 1000, sc‐101145; Santa Cruz Biotechnology), OPN (dilution 1 : 1000, sc‐21742; Santa Cruz Biotechnology) and Osterix (dilution 1 : 1000, sc‐22538; Santa Cruz Biotechnology) overnight at 4 °C and then incubated with horseradish peroxidase‐labeled secondary antibodies (dilution 1 : 5000) for 50 min at room temperature. After washing with 0.05% TBST, the protein accumulation was detected by the Millipore ECL Western Blotting Detection System (EMD Millipore, Billerica, MA, USA).
7
Bone Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence was performed as previously described [28 (link)]. Cleaned bone tissues were fixed with 4% (wt/vol) ice-cold paraformaldehyde (PFA) solution for 4 h. Then, the fixed bones were washed with PBS three times to remove PFA. After that, bones were incubated in cold 0.5 M EDTA, pH 7.4–7.6, for 24 h for decalcification. The bones were washed with PBS, and then incubated in cryoprotectant (CPT) solution at 4 °C for 24 h. Then, the decalcified bones were embedded and cryosectioned for staining. We used specific antibodies to CD31 (BD Bioscience, 553370, Franklin Lakes, NJ, USA) and Osterix (Santa Cruz Biotechnology, sc-22536). 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, D9564) was used for staining of nuclei.
8
Immunofluorescence Analysis of Embryonic Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue was prepared and processed for cryosectioning and immunofluorescence as previously described (Shpargel et al., 2020 (link)). Antibodies incubated overnight at 4°C included Cleaved Caspase 3 (1:400, Cell Signaling Technology, 9661S), BrdU (1:250, Abcam, ab6326), pH3S10 (1:400, Fortis, A301-844A-M), BRD4 (1:200, Fortis, A301-985A-M), RUNX2 (1:800, Cell Signaling Technology, 12556S or 1:150, Santa Cruz Biotechnology, sc-390351), type II collagen (1:40, Developmental Studies Hybridoma Bank, II-II6B3), Osterix (1:150, Santa Cruz Biotechnology, sc-393325), COL1A1 (1:400, Cell Signaling Technology, 72026 T) and FGFR2 (1:300, Cell Signaling Technology, 23328S). BrdU was injected intraperitoneally (50 mg/kg, Sigma-Aldrich, B5002-250MG) 45 min before embryonic dissection. For BrdU detection, slides were treated with 2N HCL/PBS at 37°C for 30 min before blocking. Cells in culture were fixed in 4% paraformaldehyde/PBS at room temperature for 10 min, extracted for 5 min in 0.5% Triton X-100/PBS, and blocked with 10% goat serum in PBS before antibody incubation (30 min at 37°C). Cellular fluorescence was calculated with Fiji/ImageJ2 using background subtracted integrated fluorescent density.
9
Comprehensive Western Blot Analysis of Osteogenesis and Adipogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting assays were conducted as previously described [32 (link)]. Briefly, the proteins were separated with SDS-PAGE gel, and then the proteins were transferred to the PVDF membrane (Millipore, USA). The protein-contained membranes were incubated with primary antibodies first, including RUNX2 (1:1000, catalog no. sc-390351, Santa Cruz, USA), OSTERIX (1:800, catalog no. sc-393325, Santa Cruz, USA), Osteocalcin (OCN) (1:500, catalog no. sc-390877, Santa Cruz, USA), Osteopontin (OPN) (1:600, catalog no. sc-21742, Santa Cruz, USA), Collagen I (1:1000, catalog no. ab270993, Abcam, USA), adipocyte fatty acid binding protein 2 (AP2) (1:1000, catalog no. 3215, Cell Signaling Technology, USA), CCAAT/enhancer-binding protein α (C/EBPα) (1:600, catalog no. ab128126, Abcam, USA), fatty acid binding protein4 (FABP4) (1:1000, catalog no. 2120, Cell Signaling Technology, USA), peroxisome proliferator-activated receptor γ (PPARγ) (1:1000, catalog no. 2443, Cell Signaling Technology, USA) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000, catalog no. KM9002T, Tianjin Sungene Biotech Co. ltd, China). Then the membranes were incubated with corresponding secondary antibody (1:10000, BOSTER Biological Technology, China). Finally, image proplus was used to analyze the expression level of these proteins.
10
Evaluating Osteoblast Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To examine the treatment effects in osteoblasts on protein expression of major regulatory genes of bone formation and resorption, MG-63 osteoblastic cells were washed with cold PBS, then lysed on ice for 30 min with radio immunoprecipitation assay (RIPA) buffer. Then, protein samples (30 mg respectively) were separated on 8% sodium dodecyl sulfate (SDS) gels and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with rabbit-derived antibodies against receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG), Runt-related transcription factor 2 (RUNX-2), osterix, osteocalcin (1:1000; all from Santa Cruz Biotech, California, USA), and β-actin (1:1000; Bioworld Technology, St. Louis, Missouri, USA) after being blocked with dry skim milk-infused PBS buffer (4%) for 1 h. Following these steps, the membranes were incubated with peroxidase-conjugated secondary antibody (goat anti-rabbit or a goat anti-mouse; 1:1000; Cell Signaling Technology, Shanghai, China) for 1 h and then were visualized by enhanced chemiluminescence (ECL; Santa Cruz Biotech) using Kodak X-OMAT LS film (Eastman Kodak, Rochester, New York, USA) post PBS washing three times. All Western blot analyses in this study were repeated at least three times
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!