To examine which signalling pathways were affected by LY, BMMs were seeded in 6-well plates at a density of 5 × 105 cells/well. The cells were pre-treated with or without 0.4 μM LY for 2 h. Cells were then stimulated with 50 ng/mL RANKL for 0, 5, 10, 20, 30 or 60 min. To determine the effect of LY on NFATc1, BMMs were treated with 50 ng/mL RANKL, with or without 0.4 μM LY, for 0, 1, 3 or 5 days. Total protein was extracted from cultured cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma Aldrich, St Louis, MO, USA). Lysates were centrifuged at 12,000 × g for 15 min, and the supernatants were collected. Proteins were resolved on 10% SDS-PAGE gels and transferred by electroblotting to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% nonfat dry milk in TBST (50 mM Tris (pH 7.6), 150 mM NaCl, 0.1% Tween 20) at room temperature for 1 h and then incubated with primary antibodies overnight at 4°C. Protein bands were developed using a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Abcam, Cambridge, MA, USA), followed by detection using an electrochemical luminescence reagent (Millipore, Billerica, MA, USA). Protein bands were visualized using the LAS-4000 Science Imaging System (Fujifilm, Tokyo, Japan).
Las 4000 science imaging system
Manufactured by Fujifilm
Sourced in Japan
The LAS-4000 Science Imaging System is a high-performance imaging device designed for scientific applications. It features a CCD camera and specialized optics for capturing detailed images of samples. The system is capable of producing high-quality images and is suitable for use in various scientific research and analysis settings.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (4 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit immunoglobulin g, by Abcam (2 mentions)
Electrochemical luminescence reagent, by Merck Group (1 mentions)
Protein antibodies, by Abcam (1 mentions)
Pvdf membrane, by Abcam (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Anti hif 1α, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
7 protocols using las 4000 science imaging system
1
RANKL Signaling Pathway Regulation by LY
2
Protein Expression Analysis of Neural Progenitor Cells
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The medium was removed from the wells, and the cells were washed with phosphate-buffered saline (PBS). Human or mouse NPCs were lysed using radioimmunoprecipitation assay buffer (Beyotime, China). The protein extract was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA), and blocked with skim milk powder for 1 h. The PVDF membrane was incubated with protein antibodies (1:1000, Abcam) at 4 °C overnight (collagen II, aggrecan, SOX9, MMP3, MMP13, ADAMTS4, and ADAMTS5). After washing with Tris-buffered saline with 0.1% Tween® 20, the cells were further incubated with the secondary antibody at room temperature (25 °C) for 2 h. Protein bands were observed using the LAS-4000 Science Imaging System (Fujifilm, Tokyo, Japan) and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Investigating INF 39 Effects on Osteoclastogenesis and Osteoblastogenesis
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To determine the effect of INF 39 on c-Fos and NFATc1, BMMs were treated with 25 ng/mL M-CSF and 50 ng/mL RANKL and with 25, 50, or 100 nM INF 39 or without it for 6 days. To determine the effect of INF 39 on Runx2 and ALP, primary calvarial osteoblasts were treated with 25, 50, or 100 nM INF 39 or without it, for 7 days. Total protein was extracted from the cultured cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich). Lysates were centrifuged at 12,000 g for 10 min, and the supernatants were collected. Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% nonfat dry milk in TBST at room temperature for 1 hour and incubated with the primary antibodies overnight at 4°C. Protein bands were visualized using LAS-4000 Science Imaging System (Fujifilm, Tokyo, Japan), and the obtained images were analyzed with ImageJ software (NIH, Bethesda, MD, USA) [25 (link), 26 (link)].
4
Osteoclast Protein Expression Analysis
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Total protein was extracted from the cells by mixing with radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich), and the supernatant was collected after centrifugation at 12,000g for 15 min. The proteins were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Bio-Rad). The membrane was blocked with 5% skim milk powder in TBST (50 mm Tris, pH 7.6; 150 mM NaCl; 0.1% Tween 20) at room temperature for 1 h and incubated with the primary antibodies against osteoclast-related proteins TRAP, cathepsin K, MMP-2, and MMP-9 at 4 °C overnight (1:1,000, Abcam). Cathepsin K and TRAP are important indicators of osteoclast activity, and MMP-2 and MMP-9 reflect cell migration ability. After washing with TBST, the membrane was further incubated with the secondary antibody at room temperature for 2 h. The protein bands were visualized with an LAS-4000 Science Imaging System (Fujifilm) and analyzed with ImageJ software (National Institutes of Health).
5
Galangin Modulates Osteoclastogenesis Signaling
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The cells were pre‐treated with 12 µmol/L galangin for 1 hours. Untreated cells were used as a control. Then, BMMs were stimulated with 30 ng/mL M‐CSF and 100 ng/ml RANKL for 0, 5, 10, 20, 30 or 60 minutes, respectively. To investigate the dose‐dependent effects of galangin, BMMs were pre‐treated with different concentrations (0, 3, 6, 12 μmol/L) of galangin for 1 hours and then stimulated with RANKL for 30 minutes. To determine the effect of galangin on NFTAc1, C‐Jun and C‐Fos, BMMs were treated with 100 ng/mL RANKL with or without 6 µmol/L galangin for 3 days. Total cellular proteins were extracted from cultured cells using RIPA lysis buffer. Lysates were centrifuged at 12 000 g for 10 minutes at 4°C, and the supernatants were collected and mixed with SDS‐sampling buffer, followed by incubation at 100°C for 5 minutes. Samples were then resolved by SDS‐PAGE gels and transferred into nitrocellulose membranes via electroblotting. Membranes were blocked with 5% skim milk for 2 hours and probed with primary antibodies overnight at 4°C. Membranes were then washed and incubated with HRP‐conjugated secondary antibodies for 2 hours. Immunoreactivity detection was performed using a LAS‐4000 Science Imaging System (Fujifilm, Tokyo, Japan), and the obtained images were analysed with ImageJ.
6
Western Blot Analysis of Cellular Proteins
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RIPA lysis buffer was used to extract total protein from cells. After centrifuged the lysates, supernatants were obtained and quantified. Proteins were separated by 10% SDS-PAGE. Then they were transferred to polyvinylidene difluoride (PVDF) membranes. 5% non-fat dry milk dissolved in TBST was used to block the membranes for 1 h at room temperature. Then the membranes were incubated with specific primary antibodies at 4°C overnight. A LAS-4,000 Science Imaging System (Fujifilm, Tokyo, Japan) was used to detect protein bands and obtain images. The Image J software was used for analysis.
7
Western Blot Analysis of CA12 and HIF-1α
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Cells or ground NP tissues were incubated in RIPA buffer (Cell Signaling Technology, Boston, MA, USA) supplemented with 100 mM phenylmethanesulfonyl fluoride (PMSF) on ice, followed by centrifugation at 12 000 rpm for 15 min to isolate the supernatant. Proteins were resolved by 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad) by electroblotting. The membranes were blocked with 5% non-fat dry milk in TBST at room temperature for 1 h and then incubated with anti-CA12 (1:1000; Cell Signaling Technology), anti-HIF-1α (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-β-actin (1:2000; Santa Cruz Biotechnology) polyclonal immunoglobulin G antibodies overnight at 4 °C. Protein bands were developed using a horseradish peroxidase-conjugated goat-anti-rabbit immunoglobulin G (Abcam, Cambridge, MA, USA), followed by detection with ECL reagent (Millipore, Billerica, MA, USA). Protein bands were visualized using the LAS-4000 Science Imaging System (Fujifilm, Tokyo, Japan).
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