SMAD Reporter

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SMAD Reporter

Cignal SMAD Reporter (GFP) Kit: CCS-017G

The SMAD Reporter kit is designed to monitor the activity of TGFβ-induced signal transduction pathways in cultured cells. The kit contains transfection-ready SMAD reporter construct as well as positive and negative controls. The transforming growth factor β (TGFβ) signaling pathway is involved in many cellular processes, including cell cycle arrest, differentiation, homeostasis, and immunosuppression. TGFβ signaling induces phosphorylation and activation of the SMAD2 and SMAD3 proteins, which then form complexes with the mediator SMAD4. These SMAD complexes then translocate to the nucleus, where they activate the expression of TGFβ-responsive genes. The SMAD reporter encodes the Monster GFP gene under the control of a minimal (m)CMV promoter and tandem repeats of the SMAD transcriptional response element (TRE). We have experimentally optimized the number of response elements as well as the intervening sequence between response elements to maximize the signal to noise ratio.

Using a simple GFP reporter assay, the SMAD Reporter (GFP) can easily and rapidly:

Monitor SMAD-regulated signaling pathway activity in cells
Study chemical compounds or drugs
Analyze phenotypes of RNAi or gene over expression

See the complete transcription factor reporter assay list.

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Brief Protocol

The Cignal Reporter Assays (GFP) include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. GFP expression is quantitated using a flow cytometer, fluorescent microscope, or fluorometer. The change in the activity of each signaling pathway is determined by comparing the GFP activities in treated versus untreated transfectants. The identically treated negative control transfectants serve as a specificity control. The positive control serves as a control for transfection efficiency, by monitoring GFP expression from the constitutively expressing CMV-GFP reporter.

Performance Data

Excellent signal to noise ratio

Fluorescence Microscopy: Cignal SMAD-GFP reporter measures TGFβ signaling pathway activity

HEK293 cells were transfected with the Cignal SMAD-GFP reporter or negative control (for transfection protocol refer, to our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 24 hours of transfection, cells were treated with 25 ng/ml recombinant human transforming growth factor β 1 protein (hTGFβ1). After 18 hours of treatment, bright field and fluorescent images were taken of the cultures transfected with the negative control reporter (A and B, respectively) and the Cignal SMAD-GFP reporter (C and D, respectively).

Quantitative Fluorometry: Cignal SMAD-GFP reporter measures TGFβ signaling pathway activity

HEK293 cells were transfected with the Cignal SMAD-GFP reporter or negative control (for transfection protocol, refer to our user manual). After 16 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin). After 24 hours of transfection, cells were treated with 25 ng/ml recombinant human transforming growth factor β 1 protein (hTGFβ1). After 18 hours of treatment, medium was removed from the wells and fluorescence activity was measured using a fluorometer. The fluorescent activity present in treated and non-treated negative control wells was subtracted from the fluorescent activity in treated and non-treated Cignal SMAD-GFP reporter wells and relative fluorescence activities are expressed as arbitrary units. Experiments were done in triplicates, and the standard deviations are indicated.

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