Antibodies to GAPDH, IGF-1, IGF-1R, pho-IGF-1R, pho-IRS-1(Ser636/639), extracellular signal-regulated kinase (ERK), pho-ERK(Thr202/Tyr204), pho-AKT (Ser473), AKT, PARP, cyclin D1, cyclin D2, cyclin D3 and horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse and goat anti-rabbit) were purchased from Abcam (Cambridge, MA, USA), Millipore (Billerica, MA, USA), and Cell Signaling (Danvers, MA, USA). Recombinant mouse IGF-1, anti-human IGF-1 and anti-mouse IGF-1 enzyme-linked immunosorbent assay (ELISA) kit were obtained from R&D Systems (Minneapolis, MN, USA). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was from Promega (Madison, WI, USA).
Cyclin d3
Manufactured by Abcam
Sourced in United States
Cyclin D3 is a protein that plays a key role in the regulation of the cell cycle. It is involved in the transition from the G1 phase to the S phase of the cell cycle. Cyclin D3 forms a complex with cyclin-dependent kinases (CDKs), which phosphorylate and inactivate the retinoblastoma protein (Rb), allowing the cell to progress through the cell cycle.
Automatically generated - may contain errors
Lab products found in correlation
Cyclin d1, by Abcam (3 mentions)
Seramir exosome rna purification kit, by System Biosciences (2 mentions)
Igf 1, by Abcam (1 mentions)
Cyclin d2, by Abcam (1 mentions)
Igf1r, by Abcam (1 mentions)
Recombinant mouse igf 1, by R&D Systems (1 mentions)
Tnf α, by Abcam (1 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Abcam (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Tissue dissociation kit, by Miltenyi Biotec (1 mentions)
Cell cycle and apoptosis detection kit, by Beyotime (1 mentions)
Bcl 2, by Bioworld Technology (1 mentions)
Tunel kit, by Beyotime (1 mentions)
Caspase 9, by Merck Group (1 mentions)
Dot1l, by Abcam (1 mentions)
H3k79me2, by Abcam (1 mentions)
4 protocols using cyclin d3
1
Comprehensive Analysis of IGF-1 Pathway Signaling
2
Exosomal miRNA and mRNA Expression Analysis
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TRIzol (Invitrogen) was utilized to extract total RNA, and a Verso cDNA Synthesis Kit (Thermo Fisher Scientific) was used to reverse-transcribe the RNA according to the instructions of the kit. The SeraMir Exosome RNA Purification Kit (System Biosciences, Mountain View, USA) was used to extract exosomal miRNAs, and the TaqMan microRNA assay kit (Applied Biosystems, Foster City, USA) was utilized for cDNA synthesis. The StepOne Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) was used for RT-qPCR reactions. GAPDH and U6 were used for the normalization of mRNA and miRNA expressions, respectively, and the 2−ΔΔCt method was used to quantify relative expressions: IL-1β (Abcam, UK), IL-6 (Abcam, UK), TNF-α (Abcam, UK), cyclin D1 (Abcam, UK), and cyclin D3 (Abcam, UK).
3
Cell Cycle and Apoptosis Detection
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Cell cycle and apoptosis detection kit and the TUNEL kit were purchased from Beyotime biotechnology company (Beyotime, Jiangsu, China). Tissue dissociation kit was purchased from Miltenyi (Miltenyi, Bergisch Gladbach, Germany). Antibodies against ING4 (1:1000), Bax (1:1000), cyclin D1 (1:5000), cyclin D3 (1:5000), CDK2 (1:5000) and CDK4 (1:5000) were purchased from Abcam (Shanghai, China). Antibodies against Caspase 8 (1:1000), Caspase 3 (1:1000) and Bcl-2 (1:1000) were obtained from the Bioworld Company (Minnesota, USA). Caspase 9 (1:1000), XIAP (1:1000) and p21 (1:1000) antibodies were purchased from EMD Millipore Corporation (Billerica, MA, USA). PARP antibody (1:1000) was purchased from Sino Biological Inc. (Beijing, China). β-actin (1:5000) antibody was obtained from HuaAn Biotechnology Co., Ltd. (Hangzhou, China). The primers used in this study were synthesized in Generay Biotech Co.,Ltd (Shanghai, China).
4
Western Blot Analysis of Cell Lysates
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Western blot assay was performed following the protocol as previously described [24 (link), 25 (link)]. Briefly, the cells were harvested and lysed using ice-cold RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1% Nonidet P-40, and 0.5% sodium deoxycholate). Following centrifugation at 10,000×g for 15 min at 4 °C, the proteins in the supernatants were quantified by Bradford method and separated using 10% SDS-PAGE gel and electrotransferred from the gel to a nitrocellulose membrane (Merck & Co., Inc., Whitehouse Station, NJ, USA). Following blocking with 5% skimmed milk in phosphate-buffered saline, the membranes were immunoblotted with the primary antibodies, namely, CDK6 (GeneTex, San Antonio, Texas, USA), DOT1L, H3, H3K79me2, cyclin D3, and GAPDH (Abcam, Cambridge, UK) at 4 °C overnight. Specifically bound HRP-conjugated secondary antibodies were detected using an ECL detection system (ChemiDocTM XRS+ machine, Bio-Rad Laboratories). Protein levels of GAPDH and H3 were employed as loading controls. Densitometric analyses were performed using ImageJ software. Relative quantification was carried out after normalization to the band intensities of GAPDH or H3. A Mann-Whitney test was performed to assess the difference of protein expression between groups. Each experiment was performed in triplicate and repeated at least 3 times.
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