Total RNA was extracted from Splenocytes' pellets of WT and OI mice or OI mice transplanted with PBS, WT, and OI Tregs and from osteoclasts and osteoblasts from WT and OI Treg conditioned media treated cultures as well as cultures from PBS, WT, and OI Treg transplanted OI mice, using Trizol reagent (Life technologies). cDNA was generated from 1 μg total RNA using the iScript cDNA Synthesis Kit (BioRad). Quantitative real-time PCR was performed using a TaqMan Gene Expression Assays for TGF-β receptors I and II, Nuclear Factor of Activated T Cells 1 (NFATc1), Cathepsin K (CTSK), Tartrate-resistant Acid Phosphatase (TRAP), Runt-related Transcription Factor 2 (Runx-2), Alkaline Phosphatase and Osteocalcin and StepOne Real-time PCR System (Thermo Fisher). In addition, expression was quantified relative to β-Actin.
Cathepsin k
Manufactured by Thermo Fisher Scientific
Sourced in United States
Cathepsin K is a proteolytic enzyme that plays a role in the degradation of bone and cartilage matrix proteins. It is primarily expressed in osteoclasts and chondrocytes, and is involved in the normal remodeling and turnover of these tissues.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Osteocalcin, by Thermo Fisher Scientific (1 mentions)
Pristimerin, by Merck Group (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taqtm kit, by Thermo Fisher Scientific (1 mentions)
Abi 7500 analyzer, by Thermo Fisher Scientific (1 mentions)
3 protocols using cathepsin k
1
Treg-Mediated Regulation of Bone Homeostasis
2
Pristimerin and Alendronate Antiosteoporosis Study
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Pristimerin was provided by Sigma Aldrich Ltd., USA, and alendronate was provided by Hangzhou Moshadong Pharmaceutical Co. Ltd., China. ELISA kits with collagen type I fragments, osteocalcin, cathepsin K, BGP, TNF-α, and IL-1β were purchased from Thermo Fisher Scientific Ltd., USA. Antibodies were procured from Bio-Techne China Ltd., China.
3
Quantitative RT-PCR Analysis of Autophagy and Osteoclastogenesis Genes
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The total RNA was extract and purified by the TRIzol method. Synthesis of cDNA and real-time quantitative PCR (qRT-PCR) measurements were performed as described previously [21 (link)]. The pre-designed primer sequences for qRT-PCR analysis were as following:
Cathepsin K (CTSK): 5′-GGAAGAAGACTCACCAGAAGC-3′ (forward) and 5′-GTC-ATATAGCCGCCTCCACAG-3′ (reverse); Matrix metalloproteinase-9 (MMP-9):5′-CC-TGTGTGTTCCCGTTCATCT-3′ (forward) and 5′-ACCCGAATCTAGTAAGGTCGC-3′ (reverse); TRAP: 5′-GCTGGAAACCATGATCACCT-3′ (forward) and 5′-TTGAGCCAGG-ACAGCTGAGT-3′ (reverse); Atg7, 5′-GTTCGCCCCCTTTAATAGTGC-3′ (forward) and 5′-TGAACTCCAACGTCAAGCGG-3′ (reverse); Atg5, 5′-ATGCGGTTGAGG-CTCACTTTA-3′ (forward) and 5′-GGTTGATGGCCCAAAACTGG-3′ (reverse); BECN1: 5′-CTAAGGCAGGCAGGAGGATG-3′ (forward) and 5′-GCTGGCCTCAA-GAGATCCAT − 3′ (reverse); Cyclophillin A: 5′-CGAGCTCTGAGCACTGGAGA-3′ (forward) and 5′-TGG-CGTGTAAAGTCACCACC-3′ (reverse).
qRT-PCR analysis was carried out by SYBR Premix Ex TaqTM kit and using ABI7500 analyzer (Thermo, MA, USA).
Cathepsin K (CTSK): 5′-GGAAGAAGACTCACCAGAAGC-3′ (forward) and 5′-GTC-ATATAGCCGCCTCCACAG-3′ (reverse); Matrix metalloproteinase-9 (MMP-9):5′-CC-TGTGTGTTCCCGTTCATCT-3′ (forward) and 5′-ACCCGAATCTAGTAAGGTCGC-3′ (reverse); TRAP: 5′-GCTGGAAACCATGATCACCT-3′ (forward) and 5′-TTGAGCCAGG-ACAGCTGAGT-3′ (reverse); Atg7, 5′-GTTCGCCCCCTTTAATAGTGC-3′ (forward) and 5′-TGAACTCCAACGTCAAGCGG-3′ (reverse); Atg5, 5′-ATGCGGTTGAGG-CTCACTTTA-3′ (forward) and 5′-GGTTGATGGCCCAAAACTGG-3′ (reverse); BECN1: 5′-CTAAGGCAGGCAGGAGGATG-3′ (forward) and 5′-GCTGGCCTCAA-GAGATCCAT − 3′ (reverse); Cyclophillin A: 5′-CGAGCTCTGAGCACTGGAGA-3′ (forward) and 5′-TGG-CGTGTAAAGTCACCACC-3′ (reverse).
qRT-PCR analysis was carried out by SYBR Premix Ex TaqTM kit and using ABI7500 analyzer (Thermo, MA, USA).
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