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Recombinant human bmp6

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Recombinant human BMP6 is a protein product produced in a mammalian cell expression system. BMP6 is a member of the bone morphogenetic protein family and plays a role in bone and cartilage development.

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Lab products found in correlation

Ldn193189, by Merck Group (2 mentions) Human orexin a, by Fujifilm (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Ldn 212854, by Merck Group (1 mentions) Ldn 193189, by R&D Systems (1 mentions) Recombinant human tgf β1, by R&D Systems (1 mentions) Tgf β1, by R&D Systems (1 mentions) Opti mem medium, by Thermo Fisher Scientific (1 mentions) Viia7, by Thermo Fisher Scientific (1 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions) Superscript 3, by Thermo Fisher Scientific (1 mentions) Primer pairs, by Integrated DNA Technologies (1 mentions) Mem glutamax, by Thermo Fisher Scientific (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Sybr green master mix, by Roche (1 mentions) Hepg2 c3a, by American Type Culture Collection (1 mentions) Romidepsin, by Selleck Chemicals (1 mentions) Rgfp966, by Selleck Chemicals (1 mentions) Tubacin, by Merck Group (1 mentions) Pci 34051, by Selleck Chemicals (1 mentions)

Close spelling variants of this product

Recombinant BMP6 Human BMP6

12 protocols using recombinant human bmp6

1

Investigating BMP6 and TGF-β1 Signaling in HSG Cells

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HSG cells were plated at 4 × 105 cells per well in 35-mm plates, and cultured in 2 mL of DMEM/Nutrient Mixture F-12 (DMEM/F-12) with 5% FBS. After 24 h, medium was switched to low-serum medium containing DMEM/F-12 with 0.2% FBS. After a 24 h incubation, cells were treated with the following reagents for an additional 24 h: LDN-212854 (Cat# SML0965, Sigma-Aldrich Corp., The Woodlands, TX, USA) or LDN-193189 (Cat# SML0559, Sigma-Aldrich Corp.), 10 or 60 nM; recombinant human BMP6 (Cat# 507-BP-020, R&D Systems, Minneapolis, MN, USA), 6 or 25 ng/mL; and recombinant human TGF-β1 (Cat# 240-B-002, R&D Systems), 5 ng/mL. For inhibitors studies, BMP6 was added to the medium together with LDN-212854 or LDN-193189, while TGF-β1 was added during the last 45 minutes (min) of this treatment, before cells were harvested. The resuspension medium (DMSO or H2O) was used as the negative control for LDN-212854 or LDN-193189 respectively.
Yin H., Kalra L., Lai Z., Guimaro M.C., Aber L., Warner B.M., Michael D., Zhang N., Cabrera-Perez J., Karim A., Swaim W.D., Afione S., Voigt A., Nguyen C.Q., Yu P.B., Bloch D.B, & Chiorini J.A. (2020). Inhibition of bone morphogenetic protein 6 receptors ameliorates Sjögren’s syndrome in mice. Scientific Reports, 10, 2967.
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2

GNPAT Silencing in HepG2/C3A Cells

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A human liver-derived cell line, HepG2/C3A (CRL-10741, American Type Culture Collection, Manassas, VA), was used for functional studies. Cells were grown in MEM + Glutamax supplemented with 10% fetal calf serum (Life Technologies, Mulgrave, VIC, Australia). Control non-specific siRNA (si.NS) and siRNA specific for GNPAT (si.GNPAT; 10 pmol, GenePharma, Shanghai, China) were transfected into 1×105 HepG2/C3A cells in triplicate with Lipofectamine RNAimax (Life Technolgies) according to the manufacturer’s recommendations for reverse transfection at approximately 30% cell confluency. After 72 hours, RNA was isolated using Trizol (Life Technologies) and reverse transcribed with SuperScript III (Life Technologies). Quantitative PCR (qPCR) was performed on a Viia7 (Life Technologies) using SYBR green master mix (Roche, Castle Hill, NSW, Australia) for GNPAT, HAMP, ID1, and SMAD7, and expression levels were compared to the geometric means of reference genes ACTB and HPRT1 using 2−ΔCT. Primer pairs (Integrated DNA Technologies, Coralville, IA) used for quantitative PCR and GNPAT siRNA oligonucleotide sequences are shown in Table 1. For BMP6 treatment, cells were serum deprived for 6 hours in Opti-mem medium (Life Technologies), before the addition of recombinant human BMP6 (R&D Systems, Minneapolis, MN) (10 ng/mL; 4 hours) followed by harvesting.
McLaren C.E., Emond M.J., Subramaniam V.N., Phatak P.D., Barton J.C., Adams P.C., Goh J.B., McDonald C.J., Powell L.W., Gurrin L.C., Allen K.J., Nickerson D.A., Louie T., Ramm G.A., Anderson G.J, & McLaren G.D. (2015). Exome sequencing in HFE C282Y homozygous men with extreme phenotypes identifies a GNPAT variant associated with severe iron overload. Hepatology (Baltimore, Md.), 62(2), 429-439.
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3

Preparation of HDAC Inhibitors and BMPs

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PB (LC Laboratories, Woburn Massachusetts, USA) was reconstituted in DMSO and, for mouse experiments was diluted in vehicle (H2O with tween 5%, PEG 5%). Specific HDAC inhibitors were reconstituted in DMSO: Romidepsin (Selleck), RGFP966 (Selleck), LMK235 (Selleck), Tasinquimod (Selleck), Tubacin (Sigma), PCI-34051 (Selleck). Recombinant Human BMP6 (R&D systems) and Recombinant Human BMP9 (R&D systems) were reconstituted in sterile 4 mM HCl containing 0.1% bovine serum albumin. LDN193189 (Sigma) was diluted in H2O.
Pasricha S.R., Lim P.J., Duarte T.L., Casu C., Oosterhuis D., Mleczko-Sanecka K., Suciu M., Da Silva A.R., Al-Hourani K., Arezes J., McHugh K., Gooding S., Frost J.N., Wray K., Santos A., Porto G., Repapi E., Gray N., Draper S.J., Ashley N., Soilleux E., Olinga P., Muckenthaler M.U., Hughes J.R., Rivella S., Milne T.A., Armitage A.E, & Drakesmith H. (2017). Hepcidin is regulated by promoter-associated histone acetylation and HDAC3. Nature Communications, 8, 403.
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4

Antibody-Based Analysis of BMP and VEGF Signaling

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Recombinant human BMP-6, BMP-9, VEGF, bFGF and TGF-β were
purchased from R&D Systems (R&D Systems, Bio-Techne, Minneapolis,
MN, USA). Cycloheximide (CHX) was purchased from Sigma-Aldrich (Merck,
Darmstadt, Germany), and MG132 was from Peptide Institute (Osaka, Japan). The
following antibodies were used: anti-FLAG M2 (F3165; Sigma-Aldrich, Merck),
anti-DDDDK (PM020; MBL, Tokyo, Japan) which recognizes the FLAG epitope,
anti-Myc, anti-HA (HA124; Nacalai Tesque, Kyoto, Japan), anti-HA (#561; MBL),
anti-α-tubulin (AC-15; Sigma-Aldrich, Merck), anti-SMAD1 (913C1b; Bio
Matrix Research, Chiba, Japan) which recognizes both SMAD1 and SMAD5 for ChIP
assays (5 (link)), anti-phospho-SMAD1/5/8 (Cell
Signaling Technology, Danvers, MA, USA), anti-PECAM1 (clone 18; BD bioscience,
San Jose, CA, USA), anti-HIF-1α (NB100-449, Novus Biologicals,
Bio-Techne), anti-HIF-2α (NB100-122, Novus Biologicals, Bio-Techne),
anti-α-smooth muscle actin (clone 1A4; Sigma-Aldrich, Merck), and
anti-HDAC1 (clone 2E10, #05-100; Millipore, Merck). A rabbit polyclonal
anti-ATOH8 antibody was raised against residues 306-321 of human ATOH8,
CTRTLQAEGRAKKRKE.
Morikawa M., Mitani Y., Holmborn K., Kato T., Koinuma D., Maruyama J., Vasilaki E., Sawada H., Kobayashi M., Ozawa T., Morishita Y., Bessho Y., Maeda S., Ledin J., Aburatani H., Kageyama R., Maruyama K., Heldin C.H, & Miyazono K. (2019). The ALK-1/SMAD/ATOH8 axis attenuates hypoxic responses and protects against the development of pulmonary arterial hypertension. Science signaling, 12(607), eaay4430.
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5

Adrenocortical Cell Culture with Forskolin, BMP-6, and Orexin A

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Forskolin (FSK) was purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA) and recombinant human BMP-6 was purchased from R&D Systems Inc. (Minneapolis, MN, USA). Human orexin A was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The H295R human adrenocortical cell line was cultured in DMEM/F12 containing 10% FBS supplemented with insulin-transferrin-selenium (ITS-G; Thermo Fisher Scientific, Waltham, MA, USA) with 5% CO2 at 37 °C [27 (link)].
Soejima Y., Iwata N., Nishioka R., Honda M., Nakano Y., Yamamoto K., Suyama A, & Otsuka F. (2023). Interaction of Orexin and Bone Morphogenetic Proteins in Steroidogenesis by Human Adrenocortical Cells. International Journal of Molecular Sciences, 24(16), 12559.
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6

Culturing Mouse Gonadotrope Cells

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Recombinant human BMP-6 and BMP-15 were purchased from R&D Systems Inc. (Minneapolis, MN, USA). Human orexin A was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and GnRH human acetate salt was purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). LβT2 cells derived from a mouse gonadotrope cell line were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS) supplemented with penicillin-streptomycin in 12-well plates under a 5% CO2 atmosphere at 37 °C.
Soejima Y., Iwata N., Nakayama N., Hirata S., Nakano Y., Yamamoto K., Suyama A., Oguni K., Nada T., Fujisawa S, & Otsuka F. (2022). Mutual Effects of Orexin and Bone Morphogenetic Proteins on Gonadotropin Expression by Mouse Gonadotrope Cells. International Journal of Molecular Sciences, 23(17), 9782.
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7

Isolation and Culture of Granulosa Cells

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DME/F-12 cell culture medium (HyClone, Logan, UT, USA), Fetal bovine serum (HyClone, Logan, UT, USA), Trypsin (HyClone, Logan, UT, USA), FSHR immunohistochemical kit (anti-rabbit) (Xuanya Bio, Shanghai, China), PCNA Rabbit pAb (Bioworld, St. Louis, MN, USA), β-Actin Rabbit pAb (Proteintech, Wuhan, China), Anti-Rabbit IgG (H+L) (Proteintech, Wuhan, China), BeyoClick™ EdU-594 (Beyotime, Shanghai, China), Recombinant Human BMP-6 (R&D Systems, Minneapolis, MN, USA), Annexin V-FITC/PI, Protein Marker (Solarbio, Beijing, China), RNAiso, Prime Script™ RT reagent Kit with gDNA Eraser, and TB Green™ Premix Ex Taq™ Ⅲ (TaKaRa, Kusatsu, Japan).
Song S., Ding W., Yao H., Wang L., Li B., Wang Y., Tang X., Zhang Y., Huang D., Xu D, & Zhao Z. (2022). BMP6 Promotes the Secretion of 17 Beta-Estradiol and Progesterone in Goat Ovarian Granulosa Cells. Animals : an Open Access Journal from MDPI, 12(16), 2132.
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8

Modulating BMP and IL-6 Signaling

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Cells were treated with 1 or 3 μg/ml anti-BMP4, anti-BMP6, anti-BMP7, or anti-IL6 neutralizing antibodies (R&D Systems). 3 μg/ml Isotope matched anti-IgG was used as a control (R&D Systems). Human recombinant BMP6 (R&D systems), BMP4 and BMP7 (GIBCO) were used at 50 – 200 ng/ml for 24 hrs. Cells were treated with human recombinant IL6 (Peprotech) for 24 hours.
Tesfay L., Clausen K.A., Kim J.W., Hegde P., Wang X., Miller L.D., Deng Z., Blanchette N., Arvedson T., Miranti C.K., Babitt J.L., Lin H.Y., Peehl D.M., Torti F.M, & Torti S.V. (2015). Hepcidin regulation in prostate and its disruption in prostate cancer. Cancer research, 75(11), 2254-2263.
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9

Primary Hepatocyte Isolation and Culture

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Primary hepatocytes were isolated as previously described,25 cultured for 16 hours in Dulbecco's modified Eagle's medium (DMEM, Gibco) containing 10% foetal bovine serum (v/v), and then collected for experiments. Where indicated, the cells were cultured with human recombinant BMP6 (R&D Systems) and/or human holo‐Transferrin (Sigma‐Aldrich).
An P., Wang H., Wu Q., Wang J., Xia Z., He X., Wang X., Chen Y., Min J, & Wang F. (2018). Smad7 deficiency decreases iron and haemoglobin through hepcidin up‐regulation by multilayer compensatory mechanisms. Journal of Cellular and Molecular Medicine, 22(6), 3035-3044.
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10

HepG2 and Primary Hepatocyte BMP6 Signaling

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HepG2 cells were plated in 12‐well plates and cultured at 37°C in 5% CO2 with Dulbecco's modified Eagle's medium (Gibco) containing 10% heat‐inactivated fetal bovine serum (Gibco) (v/v). The cells were then transfected with siRNA targeting human GNPAT/mouse Gnpat gene or control non‐specific siRNA (30 pmol/well; TransSheep; siRNA sequences were listed in Table S2) at 70% confluence with Lipofectamine 3000 (Invitrogen). Twenty‐four hours after transfection, human recombinant BMP6 (R&D systems) was added to a final concentration of 20 ng/mL and cells were incubated for additional 12 hours. The cells were then collected for RNA extraction and quantitative real‐time PCR detection. Isolation of mouse primary hepatocytes was performed as previously described. 39 The isolated primary hepatocytes from either wild‐type or Hfe−/− mice were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The siRNA transfection and BMP6 treatment steps are comparable with HepG2 cells.
An P., Wang J., Wang H., Jiang L., Wang J., Min J, & Wang F. (2020). Gnpat does not play an essential role in systemic iron homeostasis in murine model. Journal of Cellular and Molecular Medicine, 24(7), 4118-4126.
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