ChemiScreen™ ETA Calcium-Optimized FLIPR Cell Lines

ChemiScreen™ ETA Calcium-Optimized FLIPR Cell Lines

• ChemiScreen
• Chemicon (Millipore)

2 vials

Full-length human EDNRA cDNA encoding ETA

The endothelin family of peptides are potent vasoconstrictors synthesized by endothelial cells in both constitutive and inducible pathways. The biological actions of endothelins are mediated by two GPCRs, ET_A and ET_B. ET_A is the predominant receptor on smooth muscle cells and thus is the primary mediator of the vasoconstrictor activity of endothelins in vivo. Endothelin receptors are also found in the epithelium and central nervous system (Davenport, 2002). Chemicon's cloned human ET_A-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant ET_A expression on the cell surface and contains high levels of the promiscuous G protein G_α15 to enhance coupling of the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between ET_A and its ligands.

Calcium flux assay, ligand binding assays

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This gene encodes a member of the Y family of specialized DNA polymerases. It copies undamaged DNA with a lower fidelity than other DNA-directed polymerases. However, it accurately replicates UV-damaged DNA; when thymine dimers are present, this polymerase inserts the complementary nucleotides in the newly synthesized DNA, thereby bypassing the lesion and suppressing the mutagenic effect of UV-induced DNA damage. This polymerase is thought to be involved in hypermutation during immunoglobulin class switch recombination. Mutations in this gene result in XPV, a variant type of xeroderma pigmentosum.

FUNCTION: SwissProt: Q9Y253 # DNA polymerase specifically involved in DNA repair. Plays an important role in translesion synthesis, where the normal high fidelity DNA polymerases cannot proceed and DNA synthesis stalls. Plays an important role in the repair of UV-induced pyrimidine dimers. Depending on the context, it inserts the correct base, but causes frequent base transitions and transversions. May play a role in hypermutation at immunoglobulin genes. Forms a Schiff base with 5'-deoxyribose phosphate at abasic sites, but does not have lyase activity. Targets POLI to replication foci.
COFACTOR: Divalent metal cations. Prefers magnesium, but can also use manganese.
SIZE: 713 amino acids; 78413 Da
SUBUNIT: Binds REV1L (By similarity). Binds monoubiquitinated PCNA, but not unmodified PCNA. Binds POLI.
SUBCELLULAR LOCATION: Nucleus. Note=Accumulates at replication forks after DNA damage.
DOMAIN: SwissProt: Q9Y253 The catalytic core consists of fingers, palm and thumb subdomains, but the fingers and thumb subdomains are much smaller than in high-fidelity polymerases; residues from five sequence motifs of the Y-family cluster around an active site cleft that can accommodate DNA and nucleotide substrates with relaxed geometric constraints, with consequently higher rates of misincorporation and low processivity.
DISEASE: SwissProt: Q9Y253 # Defects in POLH are the cause of xeroderma pigmentosum variant type (XPV) [MIM:278750]; also designated as XP-V. Xeroderma pigmentosum (XP) is an autosomal recessive disease due to deficient nucleotide excision repair. It is characterized by hypersensitivity of the skin to sunlight, followed by high incidence of skin cancer and frequent neurologic abnormalities. XPV shows normal nucleotide excision repair, but an exaggerated delay in recovery of replicative DNA synthesis. Most XPV patients do not develop clinical symptoms and skin neoplasias until a later age. Clinical manifestations are limited to photo-induced deterioration of the skin and eyes.
SIMILARITY: Belongs to the DNA polymerase type-Y family. & Contains 1 umuC domain.

Human

Chemicon

ETA

Cells are frozen at 2 x 10e6 cells/mL in DMEM/20% fetal bovine serum/100 U/ml penicillin and streptomycin/10% DMSO. Cell line tests negative for mycoplasma.

peptide

Place cells in liquid nitrogen immediately upon receipt. Maintain frozen in liquid nitrogen for up to 5 years. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37ºC water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing 20 mL growth media, and place in a humidified 37°C incubator with 5% CO2. After 8-24 h, cells will adhere to the plate, at which time the media should be replaced to remove residual DMSO. Cells are passaged by washing with Ca⁺⁺ and Mg⁺⁺-free HBSS (10 mL/T75), incubating with 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) for 5-10 minutes at 37°C, and rapping the side of the flask to dislodge the cells. Neutralize the trypsin by addition of 4 volumes growth media. Cells are typically passaged 1:10 with every 3-4 days, and should be passaged at least once after thawing prior to use in calcium flux assays.

Endothelin

A

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• POLH
• XP-V
• FLJ21978
• FLJ16395
• RAD30
• XPV
• RAD30A
• OTTHUMP00000039868
• EC 2.7.7.7

GPCRs

ChemiScreen™ ETA Calcium-Optimized FLIPR Cell Lines

DMEM containing 4.5 g/L glucose/10% heat inactivated fetal bovine serum/1x nonessential amino acids/10 mM HEPES/0.25 mg/ml Geneticin (G418)/100 U/ml each penicillin and streptomycin

2 x 10e6 cells/mL

SPECIFICATIONS: EC50 for calcium mobilization by Endothelin-1: ~ 7 nM
IC50 for BMS 182874 at agonist 2xEC50: 43 nM
Signal:noise at agonist E_max: 500
Z' with endothelin-1 at 2x EC50: 0.85

Chem-1

GPCR Cell Lines

Chem-1

Q9Y253

1 mL per vial

NM_006502.1