For western blot analysis, MSCs (2 × 105 cells/well) were seeded in 6-well plates and cultured in osteogenic media. Cells were treated with CoCl2 (50 μM) for 1, 3, 5 and 7 days. Cryptotanshinone (10 μM) or DMSO was added to the cells in the appropriate groups for the duration of the culture. Cell lysates were extracted on day 7 [18 (link), 22 (link)]. Total protein was estimated using the BCA Protein Assay (Thermo Scientific). Total protein (20 μg) was separated by 10% SDS-PAGE (Biotech) and transferred to polyvinylidene difluoride membrane (Roche). The membranes were blocked with 5% skimmed milk (Biosharp) absorbed in 10% tris-buffered saline with 0.1% tween 20 (TBST; Gibco) at room temperature for 1 h. Then, the membranes were incubated on a shaker for 8 h at 4 °C with one of the primary antibodies: anti-HIF-1α (Santa), anti-ALP (Abcam), anti-Osx (Abcam), anti-Runx2 (Abcam), anti-Col1α1 (Santa) and anti-GAPDH (Protech). The membranes were then incubated with secondary antibody (Abbkine) and absorbed in TBST for 1 h at room temperature. Blots were visualized, and the relative density of each blot was determined using Image J software 1.49 (NID).
Anti osx
Manufactured by Abcam
Sourced in United Kingdom, United States, Japan
Anti-OSX is a laboratory reagent that can be used to detect the presence of the OSX protein in biological samples. It is a monoclonal antibody that specifically binds to and identifies the OSX protein, which is a transcription factor involved in osteoblast differentiation and bone formation.
Automatically generated - may contain errors
Lab products found in correlation
Anti runx2, by Abcam (7 mentions)
Anti alp, by Abcam (5 mentions)
Anti gapdh, by Cell Signaling Technology (4 mentions)
Anti ocn, by Abcam (3 mentions)
Anti mef2c, by Abcam (2 mentions)
Anti gapdh, by ZenBio (2 mentions)
Anti opn, by Abcam (2 mentions)
Imagequant las 4000 mini, by GE Healthcare (2 mentions)
Anti lef1, by Abcam (2 mentions)
Anti col1a1, by Abcam (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (2 mentions)
Anti bmp2, by Abcam (2 mentions)
Bca protein assay kit, by Ipsen (2 mentions)
Anti p65, by Cell Signaling Technology (2 mentions)
Anti ocn, by ABclonal (2 mentions)
Anti pparγ, by Cell Signaling Technology (2 mentions)
Anti iκbα, by Cell Signaling Technology (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
16 protocols using anti osx
1
Evaluating Osteogenic Potential of MSCs
2
Western Blot Analysis of Osteogenic Markers
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Lysis Buffer was used to extract total proteins of MC3T3-E1 cells, and 20 μg protein of each sample were separated on polyacrylamide-SDS gels (Wako, Osaka, Japan) and transferred to PVDF membranes. The membranes were incubated with anti-Runx2 (1:500, Abcam, Tokyo, Japan), anti-OSX (1:500, Abcam, Tokyo, Japan), anti-Mef2c (1:500, Abcam, Tokyo, Japan), anti-β-actin (1:1000, Cell Signaling Technology, Danvers, MA, USA) and anti-GAPDH (1:1000, Cell Signaling Technology, Danvers, MA, USA) after blocking with 5% skim milk at 4 °C overnight. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse/rabbit IgG (1:2000; Cell Signaling Technology, Danvers, MA, USA) for 1 h. Images were visualized and captured using an ECL Plus Western Blotting Detection System (GE Healthcare, Tokyo, Japan).
3
Protein Extraction and Western Blot Analysis
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DPCs were isolated by differential digestion as described above, after which total protein was extracted and normalized according to the manufacturer’s instructions. The primary antibodies were anti-APE1 (1:1,000; abcam, USA), anti-DMP1 (1:1,000; Santa cruz, USA), anti-DSP1-H (1:1,000; Santa cruz, USA), anti-OPN (1:1000; abcam, USA), anti-ALP (1:1,000; abcam, USA), anti-OSX (1:1,000; abcam, USA), anti-Axin (1:1000; abcam, USA), anti-Lef1 (1:1000; abcam, USA), anti-non-p (active) β-catenin (1:1000; CST, USA), anti-p-GSK-3β (1:1000; CST, USA), anti-P21 (1:1000; abcam, USA), anti-cyclin-D1 (1:1,000; Santa cruz, USA) and anti-GAPDH (1:10,000; Zen, China) used as internal control. Then, the membranes were rinsed with TBST (0.1% Tween-20 in 0.01 mol/L TBS), incubated with appropriate horseradish peroxidase conjugated secondary antibodies at 1:5000 (Santa Cruz, USA) at room temperature for additional 2 h, visualized by Image Quant LAS 4000 mini (GE, UK). Densitometry analysis on the bands was performed using the NIH image J software and normalizing the data to total protein levels (Supplementary Fig. 1 . and Supplementary Fig. 2 ).
4
Immunohistochemical Analysis of Bone Markers
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Sections of jaw and femur tissues were subjected to antigen retrieval (pH 6.0, Abcam, Tokyo, Japan), followed by peroxidase blocking (DAKO, Santa Clara, CA, USA). Anti-ALP (1:100, a gift from Hokkaido University, Hokkaido, Japan), anti-Runx2 (1:125, Abcam, Tokyo, Japan), anti-OSX (1:75, Abcam, Tokyo, Japan), anti-Mef2c (1:500, Abcam, Tokyo, Japan), anti-SOST (1:50, Abcam, Tokyo, Japan), anti-IL-1β (1:100, Abcam, Tokyo, Japan) and anti–HIF–1α (1:100, Abcam, Tokyo, Japan) were used as primary antibodies. The specimens were exposed to the same DAB reaction conditions, and all images were captured using a microscope (OLYMPUS, Tokyo, Japan).
5
Western Blot Analysis of Tissue-Specific Markers
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After collected cell extract, the protein samples were separated in SDS-PAGE running buffer, and transferred onto polyvinylidene difluoride membrane. After isolation for 1
the corresponding primary antibody was incubated at 4 °C overnight: Anti-Mkx (1/200, Abcam), Anti-Tnmd (1/200, Abcam), Anti-Col1a1 (1/1000, Abcam), Anti-Osx (1/1000, Abcam), Anti-Col2a1 (1/2000, Abcam), Anti-Runx2 (1/1000, Abcam), Anti-Wnt3a (1/1000, Abcam), Anti-Wnt5a (1/1000, Abcam) and Anti-β-catenin (1/10,000, Abcam), and Anti-GAPDH antibody (1/20,000, Abcam), then incubated with horseradish peroxidase (HRP)-conjugated antibodies (Abgent, ASS3403, 1/20,000). The signal was revealed with an ECL-Detection Kit (Millipore, USA).
the corresponding primary antibody was incubated at 4 °C overnight: Anti-Mkx (1/200, Abcam), Anti-Tnmd (1/200, Abcam), Anti-Col1a1 (1/1000, Abcam), Anti-Osx (1/1000, Abcam), Anti-Col2a1 (1/2000, Abcam), Anti-Runx2 (1/1000, Abcam), Anti-Wnt3a (1/1000, Abcam), Anti-Wnt5a (1/1000, Abcam) and Anti-β-catenin (1/10,000, Abcam), and Anti-GAPDH antibody (1/20,000, Abcam), then incubated with horseradish peroxidase (HRP)-conjugated antibodies (Abgent, ASS3403, 1/20,000). The signal was revealed with an ECL-Detection Kit (Millipore, USA).
6
Protein Expression Analysis Protocol
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Total protein was extracted using RIPA lysis buffer with protease and phosphatase inhibitors (Solarbio, China) at 4 °C. Protein concentration was determined using a BCA Protein Assay Kit (EpiZyme, China). Equal amounts of protein (30 μg) were subjected to 10% (w/v) SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, USA). After blocking with 5% (w/v) BSA, the membrane was incubated with primary antibodies at 4 °C overnight and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000; 115-035-003, 111-035-003, Jackson ImmunoResearch, USA) at 25 °C for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence reagent (Millipore, USA) and the grayscale of protein bands were semi-quantified using ImageJ software.
The primary antibodies used in this study included anti-PPARγ (1:1500; #2435, Cell Signaling Technology, USA), anti-IκBα (1:1500; #4814, Cell Signaling Technology, USA), anti-p65 (1:1500; #8242, Cell Signaling Technology, USA), anti-phosphorylated p65 (p-p65; 1:1500; #3033, Cell Signaling Technology, USA), anti-BMP-2 (1:1500; ab214821, Abcam, UK), anti-OSX (1:1500; ab209484, Abcam, UK), anti-OCN (1:1000; A6205, ABclonal, China), and anti-GAPDH (1:2000; #5174, Cell Signaling Technology, USA).
The primary antibodies used in this study included anti-PPARγ (1:1500; #2435, Cell Signaling Technology, USA), anti-IκBα (1:1500; #4814, Cell Signaling Technology, USA), anti-p65 (1:1500; #8242, Cell Signaling Technology, USA), anti-phosphorylated p65 (p-p65; 1:1500; #3033, Cell Signaling Technology, USA), anti-BMP-2 (1:1500; ab214821, Abcam, UK), anti-OSX (1:1500; ab209484, Abcam, UK), anti-OCN (1:1000; A6205, ABclonal, China), and anti-GAPDH (1:2000; #5174, Cell Signaling Technology, USA).
7
Chondrocyte Protein Expression Profiling
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Eleven primary antibodies, including anti-OSX, anti-COL1A1, anti-OC, anti-MMP-1, anti-MMP-3, andanti-MMP-13, were purchased from Abcam (Cambridge, UK). After culturing for 48 h, the isolated chondrocytes were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer. The total protein concentration was determined using a bicinchoninic acid (BCA) kit (Pierce, Rockford, IL, USA). Equal amounts of protein were isolated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). The membranes were blocked with 5% skim milk in Tris-buffered saline Tween (TBST) for 1 h, and then incubated with primary antibody anti-OSX (1:1,000, ab22552, Abcam), anti-COL1A1 (1:1,000, ab34710, Abcam), anti-OC (1:500, ab93876, Abcam), anti-MMP-1 (1:1,000, ab137332, Abcam), anti-MMP-3 (1:500, ab53015, Abcam), and anti-MMP-13 (1:3,000, ab39012, Abcam) at 4 °C overnight. The membranes were then washed and subjected to goat anti-rabbit immunoglobulin (Ig)G horseradish peroxidase (HRP)-conjugated secondary antibodies. The blots were detected using an enhanced chemiluminescent (ECL) detection kit (Pierce, Rockford, IL, USA). Actin was used as a loading control. The band densities were determined and analyzed with an automatic digital gel image analysis system Bio-Rad CFX-96 (Bio-Rad, CA, USA).
8
Protein Expression Analysis in Osteogenesis
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Protein was extracted with RIPA Lysis Buffer at 4°C. After quantification, an equal volume of cell lysate was loaded and separated on a 10% SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% nonfat milk in PBS, the membrane was incubated with primary antibodies at 4°C overnight (anti-Foxc1 (1/1000; CST, USA), anti-Runx2 (1/2000; Abcam, USA), anti-Osx (1/800; Abcam, USA), anti-Ocn (1/1000; Abcam, USA), and anti-GAPDH (1/2000; CST, USA). The membrane was washed with Tris-buffered saline plus 0.1% Tween 20 (TBST), and incubated with fluorescent secondary antibodies (anti-rabbit IgG and anti-rat IgG, 1/10000; CST, USA). The blot was scanned by Odyssey Infrared Imaging System (USA).
9
Western Blot Analysis of miR-920 Regulation
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Total protein was extracted from hBMSCs transfected with miR-920 mimic, inhibitor, or NC using RIPA buffer and protease inhibitors at a volume ratio of 100:1. Then, the same amount of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred to cellulose nitrate membranes and washed with TBST with 5% skim milk at room temperature for 1 h. Next, the membranes were incubated with the following primary antibodies: anti-HOXA7 (1:2000, ProteinTech), anti-ALP (1:1000, Abcam), anti-OSX (1:1000, Abcam), anti-p-p38 (1:1000, Cell Signaling Technology), anti-p38 (1:1000, Cell Signaling Technology), anti-p-JNK (1:1000, Cell Signaling Technology), anti-JNK (1:1000, Cell Signaling Technology), and anti-GAPDH (1:3000, ProteinTech) at 4 °C overnight. The membranes were warmed up to room temperature, washed with TBST 3 times, incubated with secondary antibody (1:5000; ProteinTech) for 2 h, washed with TBST 3 times, and then ECL chemiluminescence and development were performed in the dark. Amersham Imager 600 (GE Healthcare) was used to observe the bands on the PVDF membrane.
10
Multiparametric Flow Cytometry of Adipose Tissue
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