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CYTOKINE SIGNALING 4
HEK-Blue TGF-b Cells - TGF-b Reporter Cells
➥ Monitor the TGF-b/Smad pathway
➥ Detection of human TGF-b
Background
Tumor growth factor-beta (TGF-b) belongs to a family of structurally
related cytokines that regulate a plethora of cellular functions, such as
proliferation, apoptosis, differentiation and migration. TGF-b binds to a
type II receptor which recruits and activates a type I receptor. The type I
receptor then phosphorylates receptor-regulated Smads (R-Smads), such
as Smad2 and Smad3, which associate with Smad4. R-Smad/Smad4
complexes accumulate in the nucleus where they regulate the
transcription of target genes.
Description
HEK-Blue TGF-b cells allow the detection of bioactive TGF-b by
monitoring the activation of the TGF-b/Smad pathway. They were
generated by stable transfection of HEK293 cells with the human TGFBRI,
Smad3 and Smad4 genes. They further express a SEAP reporter gene
under the control of the b-globin minimal promoter fused to three
Smad3/4-binding elements (SBE).
Stimulation of HEK-Blue TGF-b cells with TGF-b induces the activation
of the TGF-b/Smad signaling pathway leading to the formation of a
Smad3/Smad4 complex. The heterocomplex enters the nucleus and binds
SBE sites inducing the production of SEAP (figure 1). The quantity of SEAP
secreted in the supernatant can be easily assessed using QUANTI-Blue
(figure 2). TGF-b-mediated SEAP production can be blocked using a
neutralizing antibody, such as anti-hTGF-b-IgA (see page 104).
• Detection range for TGF-b: 0.1 - 10 ng/ml
HEK-Blue TGF-b cells are resistant to blasticidin, hygromycin and
Zeocin .
Contents and Storage
HEK-Blue TGF-b cells are grown in DMEM medium, 2 mM L-glutamine,
10% FBS supplemented with 30 μg/ml blasticidin, 200 µg/ml HygroGold
(ultrapure hygromycin) and 100 µg/ml Zeocin . Cells are provided frozen
in a cryotube containing 5-7 x 10 6 cells and supplied with 100 µl of
blasticidin at 10 mg/ml, 100 µl of HygroGold at 100 mg/ml, 100 µl of
Zeocin at 100 mg/ml and 1 pouch of QUANTI-Blue . Cells are shipped
on dry ice.
HEK-Blue TGF-b cells are also available in a kit, see below:
HEK-Blue CD40L Kit
• HEK-Blue TGF-b Cells 5-7 x 10 6 cells
• Anti-hTGF-b-IgA antibody 100 μg
• QUANTI-Blue 1 pouch (100 ml)
• Normocin 1 ml (50 mg/ml)
• Blasticidin 100 μl (10 mg/ml)
• HygroGold 100 μl (100 mg/ml)
• Zeocin 100 μl (100 mg/ml)
114 www.invivogen.com/cytokine-sensor-cells
Figure 1: TGF-b-induced Smad signaling pathway
Figure 2: Stimulation of HEK-Blue TGF-b cells by human TGF-b was assessed by
measuring the levels of SEAP using QUANTI-Blue .
PRODUCT QUANTITY CAT. CODE
HEK-Blue TGF-b Cells 5-7 x 10 6 cells hkb-tgfb
HEK-Blue TGF-b Kit 1 kit hkb-tgfb-kit