Cat. No：GM-032AS008
Product：H_GARP Latent TGFβ-αvβ6 Blockade Assay
Cat. No：GM-032AS008
Product：H_GARP Latent TGFβ-αvβ6 Blockade Assay
H_GARP Latent TGFB1 Reporter HEK-293 Cell Line
Cell Growth Medium：DMEM+10% FBS+1% P.S+4 μg/mL Blasticidin+150 μg/mL Bleomycin+400 μg/mL G418+125 μg/mL Hygromycin+0.75 μg/mL Puromycin
Cell Freezing Medium：90% FBS+10% DMSO
H_αvβ6 HEK-293 Cell Line
Cell Growth Medium：DMEM+10% FBS+1% P.S+150 μg/mL Bleomycin+125 μg/mL Hygromycin
Cell Freezing Medium：90% FBS+10% DMSO
Assay Buffer：DMEM+10% FBS+1% P.S
Regulatory T cells (Tregs) play a role in suppressing excessive immune responses and preventing autoimmune diseases by producing transforming growth factor-beta (TGF-β). The proprotein encoded by transforming growth factor-beta (TGF-β), known as latency-associated peptide (LAP), can be cleaved by the furin enzyme to release TGF-β peptide. Subsequently, it forms a complex by binding with latent TGF-beta binding proteins (LTBPs). Moreover, it can also interact with glycoprotein A repetitions predominant (GARP) highly expressed on the surface of Tregs to release inactive TGF-β into the extracellular matrix (ECM) or accumulate on the cell surface, with a stronger binding capacity than the former. Following this, the GARP-proTGFβ complex is activated by integrins to form active TGF-β. Integrin αvβ6, a transmembrane heterodimer composed of α and β subunits through non-covalent bonds, binds LAP through the high-affinity RGD LXXL binding site on its protein structure, leading to the activation of latent TGF-β.
The H_GARP Latent TGFβ-αvβ6 Blockade Assay is a mechanism of action-based bioassay that can be used to assess the efficacy and stability of antibodies and other biological agents that block the interaction between H_GARP Latent TGFβ and αvβ6. This assay involves two genetically engineered cell lines: the H_GARP Latent TGFB1 Reporter HEK-293 Cell Line, which stably expresses human GARP gene, Latent TGFβ, and a TGF-β-induced luciferase reporter gene in HEK-293 cells; and the H_αvβ6 HEK-293 Cell Line, which expresses human αvβ6 in HEK-293 cells. When these two cell lines are co-cultured, TGF-β is cleaved by αvβ6 into the culture medium, binds to TβRI and TβRII receptors, and activates transcription factor-mediated expression of the luciferase gene. Addition of blocking antibodies against GARP/αvβ6 inhibits the activation of TGF-β and its downstream signaling pathways. The extent of the impact of the blocking antibodies on the signaling pathway can be assessed by measuring the fluorescent signal.
Cat. No：GM-032AS008
Product：H_GARP Latent TGFβ-αvβ6 Blockade Assay
H_GARP Latent TGFB1 Reporter HEK-293 Cell Line
Cell Growth Medium：DMEM+10% FBS+1% P.S+4 μg/mL Blasticidin+150 μg/mL Bleomycin+400 μg/mL G418+125 μg/mL Hygromycin+0.75 μg/mL Puromycin
Cell Freezing Medium：90% FBS+10% DMSO
H_αvβ6 HEK-293 Cell Line
Cell Growth Medium：DMEM+10% FBS+1% P.S+150 μg/mL Bleomycin+125 μg/mL Hygromycin
Cell Freezing Medium：90% FBS+10% DMSO
Assay Buffer：DMEM+10% FBS+1% P.S
Regulatory T cells (Tregs) play a role in suppressing excessive immune responses and preventing autoimmune diseases by producing transforming growth factor-beta (TGF-β). The proprotein encoded by transforming growth factor-beta (TGF-β), known as latency-associated peptide (LAP), can be cleaved by the furin enzyme to release TGF-β peptide. Subsequently, it forms a complex by binding with latent TGF-beta binding proteins (LTBPs). Moreover, it can also interact with glycoprotein A repetitions predominant (GARP) highly expressed on the surface of Tregs to release inactive TGF-β into the extracellular matrix (ECM) or accumulate on the cell surface, with a stronger binding capacity than the former. Following this, the GARP-proTGFβ complex is activated by integrins to form active TGF-β. Integrin αvβ6, a transmembrane heterodimer composed of α and β subunits through non-covalent bonds, binds LAP through the high-affinity RGD LXXL binding site on its protein structure, leading to the activation of latent TGF-β.
The H_GARP Latent TGFβ-αvβ6 Blockade Assay is a mechanism of action-based bioassay that can be used to assess the efficacy and stability of antibodies and other biological agents that block the interaction between H_GARP Latent TGFβ and αvβ6. This assay involves two genetically engineered cell lines: the H_GARP Latent TGFB1 Reporter HEK-293 Cell Line, which stably expresses human GARP gene, Latent TGFβ, and a TGF-β-induced luciferase reporter gene in HEK-293 cells; and the H_αvβ6 HEK-293 Cell Line, which expresses human αvβ6 in HEK-293 cells. When these two cell lines are co-cultured, TGF-β is cleaved by αvβ6 into the culture medium, binds to TβRI and TβRII receptors, and activates transcription factor-mediated expression of the luciferase gene. Addition of blocking antibodies against GARP/αvβ6 inhibits the activation of TGF-β and its downstream signaling pathways. The extent of the impact of the blocking antibodies on the signaling pathway can be assessed by measuring the fluorescent signal.
