Cell Signaling Technology

Product Pathways - Growth Factors/Cytokines

Human Latent Transforming Growth Factor β1 (hLatent TGF-β1) #5154

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Source

Recombinant human latent TGF-β1 (hLatent TGF-β1) Leu30-Ser390 (Accession #P01137) was expressed in human 293 cells at Cell Signaling Technology.

Molecular Characterization

Recombinant hLatent TGF-β1 contains no "tags" and the nonglycosylated small latent TGF-β1 complex has a calculated MW of 41,251. DTT-reduced protein migrates as 40 and 13 kDa polypeptides, and the non-reduced cystine-linked homodimers migrate as 80 and 25 kDa proteins. The expected amino-terminal ALDTN of recombinant hTGF-β1 and the expected amino-terminal LSTSK of recombinant latency-associated peptide (LAP) were verified by amino acid sequencing.

Purity

>98% as determined by SDS-PAGE of 6 μg reduced (+) and non-reduced (-) recombinant hLatent TGF-β1. All lots are greater than 98% pure.

Bioactivity

The bioactivity of recombinant hLatent TGF-β1 was determined by assessing inhibition of IL-4 induced HT-2 cell proliferation. The ED50 of each lot is between 4-10 ng/ml and 0.2-0.8 ng/ml after acid activation.

Coomassie Gel

Coomassie Gel

The purity of recombinant hLatent TGF-β1 was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hLatent TGF-β1 and staining overnight with Coomassie Blue.

Bioactivity

Bioactivity

The inhibition of IL-4 induced proliferation in HT-2 cells treated with increasing concentrations of hLatent TGF-β1 or acid-activated hLatent TGF-β1 was assessed. After 48 hour treatment with hLatent TGF-β1, cells were incubated with a chemiluminescent cell viability reagent and the relative light units (RLU) were determined.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, untreated or treated with the hLatent TGF-β1 for 25 minutes, using Phospho-Smad2 (Ser465/467) (138D4) Rabbit mAb #3108 (upper) and Smad2 (86F7) Rabbit mAb #3122 (lower).


Endotoxin

Less than 0.01 ng endotoxin/1 μg hLatent TGF-β1.

Formulation

With carrier: A 0.22 μm filtered solution of 0.25 mg/ml hLatent TGF-β1 in PBS, pH 7.2 and 25% (v/v) glycerol containing 20 μg BSA per 1 μg hLatent TGF-β1. Carrier free: A 0.22 μm filtered solution of 0.25 mg/ml hLatent TGF-β1 in PBS, pH 7.2 and 25% (v/v) glycerol.

Background

Latent TGF-β1 is a complex of two proteins, latency associated protein (LAP) and TGF-β1, which is derived from cleavage of a common 75 kDa precursor protein (1). The LAP protein spatially and temporally regulates TGF-β1 activity by sequestering TGF-β1 in the extracellular matrix in conjunction with latent TGF-β1 binding proteins (LTBP)(1). The release of TGF-β1 is activated by a number of stimuli including proteases, thrombospondin-1, reactive oxygen species, and some integrins (1). Active TGF-β1 binds to TβRII homodimer, which then complexes with TβRI homodimer (2,3). The oligomeric receptor complex phosphorylates subsets of the Smad proteins that then act to induce or repress a number of target genes (3-5). TGF-β1 binding can also activate the Erk2, p38, and Jnk pathways via TAK1 (5). Active TGF-β1 activities include proliferation, angiogenesis, and promotion or inhibition of many immune events (2,4,5). Latent TGF-β1 is present on the surface of regulatory T cells in association with GARP and may contribute directly to their immunosuppressive activity (6,7).

  1. Annes, J.P. et al. (2003) J Cell Sci 116, 217-24.
  2. Bierie, B. and Moses, H.L. (2006) Nat Rev Cancer 6, 506-20.
  3. Moustakas, A. and Heldin, C.H. (2009) Development 136, 3699-714.
  4. Siegel, P.M. and Massagué, J. (2003) Nat Rev Cancer 3, 807-21.
  5. Tian, M. and Schiemann, W.P. (2009) Future Oncol 5, 259-71.
  6. Tran, D.Q. et al. (2009) Proc Natl Acad Sci U S A 106, 13445-50.
  7. Stockis, J. et al. (2009) Eur J Immunol 39, 3315-22.

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