TGF-β Reporter HEK 293 Cells

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TGF-β Reporter Cells

Signaling pathway in HEK-Blue™ TGF-β cells

HEK-Blue™ TGF-β cells were engineered from the human embryonic kidney HEK293 cell line to detect bioactive human and murine transforming growth factor-beta (TGF-β) by monitoring the activation of the Smad pathway. In addition, these cells can be used for screening antibodies or small molecule inhibitors targeting the TGF-β pathway.

TGF-β regulates numerous cellular functions, such as cell proliferation, apoptosis, differentiation, and migration [1,2].

 More details

Cell line description

HEK-Blue™ TGF-β cells were generated by stable transfection of a Smad-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of TGF-β to its receptor triggers a signaling cascade leading to the activation and formation of a Smad2/Smad3/Smad5 complex. The heterocomplex enters the nucleus and binds Smad binding elements (SBEs) in the SEAP minimal promoter, thereby inducing production of the reporter. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent.

HEK-Blue™ TGF-β cells detect human (h) and murine (m) TGF-β (see figures). Of note, these cells respond to hTGF-β1, hTGF-β2, and hTGF-β3 isoforms. They are not responsive to hTNF-α and hIL-1β (see figures).

Key features

• Fully functional TGF-β signaling pathway
• Readily assessable Smad-inducible SEAP reporter activity
• Strong response to human (h) and murine (m) TGF-β

Applications

• Detection and quantification of human and murine TGF-β activity
• Screening of anti-TGF-β and anti-TGF-βRI/TGF-βRII antibodies
• Screening of small molecule inhibitors of the TGF-β pathway

References:

1. Travis MA. & Sheppard D., 2014. TGF-β activation and function in immunity. Annu Rev Immunol. 32:51-82.
2. Taylor AW., 2009. Review of the activation of TGF-beta in immunity. J Leukoc Biol. 85(1):29-33.

Figures

Dose-response of HEK-Blue™ TGF-β cells to recombinant TGF-β. Cells were stimulated with increasing concentrations of recombinant human TGF-β1 (hTGF-β1) and murine TGF-β1 (mTGF-β1). After overnight incubation, the TGF-β/Smad response was determined using QUANTI‑Blue™ Solution, a SEAP detection reagent. The optical density (OD) at 630 nm is shown as mean ± SEM.

Response of HEK-Blue™ TGF-β cells to a panel of cytokines. Cells were stimulated with various human and murine recombinant cytokines: 0.9 ng/ml of hTGF-β1, hTGF-β2, hTGF-β3, or mTGF-β, and 100 ng/ml hTNF-α or hIL-1β. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™ Solution. The optical density (OD) at 630 nm is shown as mean ± SEM.

Dose-dependent inhibition of HEK-Blue™ TGF-β cell response using Anti-TGF-β-IgG. A serial dilution of Anti-TGF-β was incubated with 3 ng/ml of hTGF-β1 for 30 minutes prior to the addition of HEK‑Blue™ TGF-β cells. After overnight incubation, the TGF-β/Smad response was determined using QUANTI‑Blue™ Solution. The optical density (OD) at 630 nm is shown as mean ± SEM.

Specifications

Antibiotic resistance: Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2-4 mM L-glutamine, 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™

Specificity: human and murine TGF-β

Detection range: 

• 10 pg/ml - 10 ng/ml for hTGF-β
• 10 pg/ml - 10 ng/ml for mTGF-β

Quality Control:

• SEAP reporter activity in response to human and murine TGF-β has been validated.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

This product is covered by a Limited Use License (See Terms and Conditions).

Contents

• 3-7 x 10⁶ HEK-Blue™ TGF-β cells in a cryovial or shipping flask
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml)
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)

Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

Details

Tumor growth factor-beta (TGF-β) belongs to a family of structurally related cytokines that regulate a plethora of cellular functions, such as proliferation, apoptosis, differentiation, and migration [1,2]. TGF-β exists in at least three isoforms; TGF-β1, TGF-β2, and TGF-β3. In the immune system, TGF-β1 is the predominant isoform [1]. It is produced by many cell types, including macrophages, in a latent form that is bound to two other polypeptides, latent TGF-β1 binding protein (LTBP) and latency-associated peptide (LAP). Upon cleavage of these proproteins, the mature TGF-β1 is released. This mature protein can bind its cell surface receptors and initiate signaling.

TGF-β binds to type II receptors (TβR-II) which recruit and activate type I receptors (TβR-I). The active ligand-heterotetrameric receptor complex signals through downstream transcriptional factors named Smads, including Smad2, Smad3, and Smad4.
Smad complexes translocate into the nucleus where they regulate the transcription of target genes, which contain one or more Smad binding elements (SBEs) in their promoter region [3]. Perturbations in TGF-β signaling affect immune homeostasis and tolerance, leading to inflammatory diseases and tumor immune evasion [3].

1.  Travis MA. & Sheppard D., 2014. TGF-β activation and function in immunity. Annu Rev Immunol. 32:51-82.
2. Taylor AW., 2009. Review of the activation of TGF-beta in immunity. J Leukoc Biol. 85(1):29-33.
3. Battle E. & Massagué J., 2019. Transforming Growth Factor-beta Signaling in Immunity and Cancer. Immunity. 50(4):924.

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