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LanthaScreen™ STAT3 GripTite™ Cell Line

The JAK/STAT3 signaling pathway is known to be activated by cytokines such as IL-6. In this pathway, binding of IL-6 to its cell-surface receptors results in the activation of JAKs, which in turn phosphoactivate STAT3 proteins at a specific tyrosine residue (Y705). LanthaScreen® STAT3 GripTite™ is a human cell line that constitutively expresses a GFP-STAT3 fusion protein. The JAK/STAT signaling pathway is known to be functionally intact in this cell line; therefore, the GFP-STAT3 fusion protein serves as a substrate for IL-6-inducible phosphorylation. Using this cell line, a lytic immunoassay has been developed in which the phosphorylation state of GFP-STAT3 is detected in cell lysates using a terbium-labeled anti-pY705-STAT3 antibody in a time-resolved FRET (TR-FRET) readout.

The GFP-STAT3 DNA expression construct was transfected into the GripTite™ HEK 293 cell line using Lipofectamine™ 2000 transfection reagent, and the transfected cells were selected with blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as a sorting marker. The assay utilizing this cell line is validated for EC50 and Z’-factors under optimized conditions using IL-6 as a ligand for JAK-mediated GFP-STAT3 phosphorylation. This assay has also been tested under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance, and assay equilibration time. An alternate "mix-and-read" assay format is also described.

Phospho-STAT3 (Ser727) Polyclonal Antibody (Invitrogen™)

Phospho-STAT3 (Ser727) Polyclonal Antibody for Western Blot

STAT3 (Total/Phospho) Multispecies InstantOne™ ELISA Kit (Invitrogen™)

STAT3 (Total/Phospho) Human InstantOne™ ELISA Kit for ELISA

Phospho-STAT3 (Tyr705) Antibody (3H16L22), ABfinity™ Rabbit Monoclonal (Invitrogen™)

Phospho-STAT3 (Tyr705) Monoclonal Antibody for Western Blot, ELISA

Phospho-STAT3 (Ser727) Polyclonal Antibody (Invitrogen™)

Phospho-STAT3 (Ser727) Polyclonal Antibody for Western Blot, IHC (P), ChIP

STAT3 Multispecies In-Cell ELISA Kit, Near Infrared (Invitrogen™)

STAT3 Multispecies In-Cell ELISA Kit, Near Infrared for ELISA

Phospho-STAT3 (Tyr705) Antibody (3HCLC), ABfinity™ Rabbit Oligoclonal (Invitrogen™)

Phospho-STAT3 (Tyr705) Oligoclonal Antibody for Western Blot, IHC (P), ChIP

GFP-STAT3

STAT3 expressed as a fusion with GFP (Green Fluorescent Protein) to act as a physiological substrate for the Janus family of tyrosine kinases.

For use in combination with the LanthaScreen® Tb-anti-STAT3 [pTyr705] Antibody (PV4846) to evaluate inhibitors in medium to high throughput screening applications.

STAT3 (Phospho) [pY705] Multispecies ELISA Kit (Invitrogen™)

STAT3 (Phospho) [pY705] Multispecies ELISA Kit for ELISA

STAT3 Polyclonal Antibody (Invitrogen™)

STAT3 Polyclonal Antibody for Western Blot

Phospho-STAT3 (Tyr705) Monoclonal Antibody (LUVNKLA), APC, eBioscience™ (Invitrogen™)

Phospho-STAT3 (Tyr705) Monoclonal Antibody for Flow

Phospho-STAT3 (Tyr705) Monoclonal Antibody (LUVNKLA), PE, eBioscience™ (Invitrogen™)

Phospho-STAT3 (Tyr705) Monoclonal Antibody for Flow

Phospho-STAT3 (Tyr705) Monoclonal Antibody (LUVNKLA), eFluor 450, eBioscience™ (Invitrogen™)

Phospho-STAT3 (Tyr705) Monoclonal Antibody for Flow

Phospho-STAT3 (Tyr705) Monoclonal Antibody (LUVNKLA), PE-Cyanine7, eBioscience™ (Invitrogen™)

Phospho-STAT3 (Tyr705) Monoclonal Antibody for Flow

STAT3 Antibody (3HCLC), ABfinity™ Rabbit Oligoclonal (Invitrogen™)

STAT3 Oligoclonal Antibody for Western Blot, IF, ICC, IHC (P), ChIP, ELISA