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Keratin17

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Keratin17 is an antibody product offered by Cell Signaling Technology. It is used in research applications to detect the Keratin 17 protein. Keratin 17 is a member of the keratin family of intermediate filament proteins, which play a structural role in cells.

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Lab products found in correlation

Collagenase 1, by Thermo Fisher Scientific (3 mentions) Ertr7, by Santa Cruz Biotechnology (2 mentions) Er tr7, by Abcam (2 mentions) Caspase 3, by Cell Signaling Technology (1 mentions) P smad1 5 8, by Cell Signaling Technology (1 mentions) β catenin, by Merck Group (1 mentions) Keratin 10, by Fortrea (1 mentions) Keratin 14, by Fortrea (1 mentions) Keratin 14, by Novus Biologicals (1 mentions) Goat anti mouse hrp, by Santa Cruz Biotechnology (1 mentions) Goat anti rabbit hrp, by Santa Cruz Biotechnology (1 mentions) Tris hcl ready gel, by Bio-Rad (1 mentions) Gapdh, by Merck Group (1 mentions) Keratin 5, by Fortrea (1 mentions)

7 protocols using keratin17

1

Immunostaining with Diverse Antibodies

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The following antibodies were used for immunostainings. β-catenin (Sigma, sigmaaldrich.com), ABC (Millipore, Billerica, MA, Millipore. com), CD34 (BD Pharmingen, San Jose, CA, BDbioSciences.com), Keratin14 (Covance, Covance.com), Keratin 10 (Covance), AE13 (Abcam), Keratin17 (gift of P. Coulombe), pSmad 1/5/8 (Cell signaling, Boston, MA, cellsignal.com), NfatC1 (Santa Cruz Bio, Dallas, Tx, scbt. com), NFKB p50 (Santa Cruz Bio), and Caspase 3 (Cell Signaling). Sections were stained using standard immunohistochemical staining procedures.
Yucel G., Van Arnam J., Means P.C., Huntzicker E., Altindag B., Lara M.F., Yuan J., Kuo C, & Oro A.E. (2014). Partial Proteasome Inhibitors Induce Hair Follicle Growth by Stabilizing β-catenin. Stem cells (Dayton, Ohio), 32(1), 85-92.
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2

Isolation and culture of prostate fibroblasts

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The prostates were collected from 8-to-10-week-old wild-type and NOX4−/− C57BL/6 mice. Prostates were then minced, and digested by 5 mg/ml Gibco® Collagenase Type I (cat. no. 17100017; Thermo Fisher Scientific Inc., Rockford, IL) for 30 min at 37°C (24 ). Tissue fragments were then cultured for 2–3 weeks in Dulbecco's minimal essential media (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% nonessential amino acids with or without 30 μM MnTE-2-PyP (a gift from Dr. James Crapo, National Jewish Health, Denver, CO; for wild-type cells only). After five days of culture, all the cells were fibroblasts. Purity of the cells was determined by ER-TR7 (cat. no. 73355; Santa Cruz Biotechnology® Inc., Dallas, TX), a fibroblast marker, and by Keratin 17 (cat. no. 4543; Cell Signaling Technology®, Danvers, MA), an epithelial cell marker. All experiments were repeated in triplicate using primary fibroblast cells collected from prostates of different mice.
Chatterjee A., Kosmacek E.A, & Oberley-Deegan R.E. (2017). MnTE-2-PyP Treatment, or NOX4 Inhibition, Protects against Radiation-Induced Damage in Mouse Primary Prostate Fibroblasts by Inhibiting the TGF-Beta 1 Signaling Pathway. Radiation research, 187(3), 367-381.
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3

Western Blot Analysis of Cell Lysates

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Western blotting was performed on up to 20 μg of the cell lystes that were used for proteomic analysis (above). Proteins were separated by SDS-PAGE using a 4–15% Tris-HCl Ready Gel (Bio-Rad). The following primary antibodies were used at 1:1000 dilution in 5% milk, including: GAPDH (Millipore MAB374), RSPH4A (Sigma-Aldrich HPA031196), repetin (Sigma-Aldrich HPA030483), TGM1 (Sigma-Aldrich HPA040171), keratin 5 (Covance PRB160P), keratin 6 (Thermo PA5–28235), keratin 14 (Novus 34270) and keratin 17 (Cell Signaling 12509). The following secondary antibodies were used at 1:3000 dilution in 5% milk: goat anti-rabbit HRP (Santa Cruz Biotech sc-2004) and goat anti-mouse HRP (Santa Cruz Biotech sc-2005).
Foster M.W., Gwinn W.M., Kelly F.L., Brass D.M., Valente A.M., Moseley M.A., Thompson J.W., Morgan D.L, & Palmer S.M. (2017). Proteomic analysis of primary human airway epithelial cells exposed to the respiratory toxicant diacetyl. Journal of proteome research, 16(2), 538-549.
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4

Isolation and Culture of Mouse Prostate Fibroblasts

Cited 1 time
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Prostates were collected from six to eight-week-old C57BL/6 mice [12 (link)]. After mincing the prostates, they were digested by 5 mg/mL of collagenase I (Thermo Fisher Scientific, Waltham, MA, USA, cat. 17100017) for 30 min at 37 °C [23 (link)]. Tissue fragments were then cultured for 2–3 weeks in Dulbecco’s Minimal Essential Media (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% non-essential amino acids. After 5 days of culture, all the cells were fibroblasts. The purity of the cells were determined by ERTR7 (Santa Cruz, cat. sc-73355), a fibroblast marker and Keratin17 (Cell Signaling cat. 4543) an epithelial cell marker. All experiments were repeated in triplicate using primary fibroblasts cells collected from prostates of different mice.
Chatterjee A., Zhu Y., Tong Q., Kosmacek E.A., Lichter E.Z, & Oberley-Deegan R.E. (2018). The Addition of Manganese Porphyrins during Radiation Inhibits Prostate Cancer Growth and Simultaneously Protects Normal Prostate Tissue from Radiation Damage. Antioxidants, 7(1), 21.
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5

Isolation and Culture of Mouse Prostate Fibroblasts

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Prostates were collected from C57BL/6 mice at 2 months post-radiation or in 6–8-week-old mice that were untreated. After mincing the prostates, they were digested in 5 mg/mL collagenase I (Thermo Fisher, Waltham, MA, USA, cat. 17100017) for 30 min at 37 °C [41 (link)]. Tissue fragments were then cultured for 2–3 weeks in Dulbecco’s Minimal Essential Media (DMEM), supplemented with 10% FBS, 1% penicillin/streptomycin and 1% non-essential amino acids with or without 30 μM MnTE-2-PyP (a gift from Dr. James Crapo, National Jewish Health, Denver, CO, USA). All the cells were fibroblasts after 5 days of culture. Purity of the cells was determined using ERTR7 (Abcam, cat. ab51824, Cambridge, MA, USA), a fibroblast marker and Keratin17 (Cell Signaling cat. 4543, Danvers, MA, USA), an epithelial cell marker. All experiments were repeated in triplicate using primary fibroblasts cells collected from prostates of different mice.
Shrishrimal S., Kosmacek E.A., Chatterjee A., Tyson M.J, & Oberley-Deegan R.E. (2017). The SOD Mimic, MnTE-2-PyP, Protects from Chronic Fibrosis and Inflammation in Irradiated Normal Pelvic Tissues. Antioxidants, 6(4), 87.
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6

Isolation and Culture of Mouse Prostate Fibroblasts

Cited 2 times
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Mouse primary fibroblasts were isolated as previously described8 (link),35 (link). Prostates were isolated from 6–8 week old C57BL/6J mice. Tissue was roughly minced with a scalpel then digested in 5 mg/mL collagenase I (Thermo Fisher, cat. 17100017, Waltham, MA, USA) for 30 min at 37 °C. Liberated cells and tissue fragments were then cultured for 2–3 weeks in Dulbecco’s Minimal Essential Media (DMEM), supplemented with 10% FBS, 1% penicillin/streptomycin and 1% non-essential amino acids. Cultures were maintained at 5% CO2 and 37 °C. After 5 days of culture only fibroblasts were present. Purity of the cells was determined using ERTR7 (Abcam, cat. ab51824, Cambridge, MA, USA), a fibroblast marker and Keratin17 (Cell Signaling, cat. 4543, Danvers, MA, USA), an epithelial cell marker. All experiments were repeated in triplicate using primary fibroblasts cells collected from prostates of different mice.
Kosmacek E.A, & Oberley-Deegan R.E. (2020). Adipocytes protect fibroblasts from radiation-induced damage by adiponectin secretion. Scientific Reports, 10, 12616.
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7

Isolation and Culture of Primary Mouse Colonic Fibroblasts

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Colons/rectums were isolated from 8-10 week old C57BL/6 mice and digested in 5 mg/mL collagenase I [54 ]. Cells and large tissue fragments were divided and cultured for 2-3 weeks in Dulbecco's Minimal Essential Media (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomyocin and 1% non-essential amino acids with or without 0.25 μM MnTnBuOE-2-PyP. Media and MnTnBuOE-2-PyP were replaced every three days or the cells were passaged at confluency. Cell types in the cultures were characterized using immunofluorescence for Keratin17 (Cell Signaling #4543) to detect contaminating epithelial cells and ERTR7 (Santa Cruz #sc-73355) to verify the presence of fibroblasts. Epithelial cells were not detected in any of the cultures used for experimentation. All experiments were performed in the second or third week of culturing when cell division and viability was maximal. All assays were repeated in triplicate using separate isolations of primary fibroblasts.
Kosmacek E.A., Chatterjee A., Tong Q., Lin C, & Oberley R.E. (2016). MnTnBuOE-2-PyP protects normal colorectal fibroblasts from radiation damage and simultaneously enhances radio/chemotherapeutic killing of colorectal cancer cells. Oncotarget, 7(23), 34532-34545.
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