Biopath Online Interferon Pathway

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New pathway web pages
Whether your Interferon pathway research involves basic research tools, cell-based assays, or comprehensive screening services, Invitrogen has solutions for you. See our new Interferon pathway web page for more information.

Interferon Pathway
 New Interferon pathway web page
Empower your research today using Invitrogen’s comprehensive portfolio of products and services to investigate the Interferon pathway—everything from high-quality reagents for basic research and assay development to validated biochemical and cell-based assays, and world-class profiling and screening services.

See our portfolio of Interferon pathway–associated reagents at www.invitrogen.com/IFN.

Enzyme-linked immunosorbent assays (ELISA) are an easy and unbiased method traditonally employed to measure interferon (IFN) proteins from serum, plasma, or tissue culture supernatant. Now, using the same method, you can also measure the associated activated or phosphorylated proteins within the cell after interferon stimulation. Among many phosphoELISA™ assays available to study the interferon pathway, Invitrogen offers kits for JAK2 [pYpY1007/1008], STAT1 [pY701], Akt [pS473], p38 MAPK [pTpY180/182], CREB [pS133], and 4E-BP1 [pT46] protein measurements.

Effect of Interferon-y treatment on STAT1 phosphorylation in HeLa cells Figure 1—Effect of IFN treatment on STAT1 phosphorylation in HeLa cells. Cells were treated with IFN  at varying concentrations (0.001 to 20 ng/ml) for 20 minutes then lysed. The level of phosphorylation at Tyr 701 increases with dosage of IFN . The results correlated very well with western blot analysis of the same samples (inset). Total STAT1 levels remain constant, while phosphorylation levels increase with IFN  dose. {edits to above image: remove title. y-axis: remove capitalization in "units/ml". x-axis: lower-case "l" in "ml".}


ELISA kits are an easy way to get reliable results in 4 hours. The kits offer a low detection limit and high consistency, and come ready to use with all the necessary reagents, including recombinant standards for quantitative results.

ELISA kits are rigorously validated to ensure excellent quality. Validation studies include experiments to verify parallelism between calibrated standards and natural samples, peptide competition and cross-reactivity tests for specificity, and stimulation experiments to ensure correct activation patterns. Specifications include excellent precision (<10% CV), lot-to-lot consistency (<20% CV), recovery (85–108%), and sensitivity (at least 2X more sensitive than western blots).   

Two complementary cellular approaches to analyze JAK/STAT pathway activation by interferons have been developed and validated for use in high-throughput screening (HTS) applications. 

LanthaScreen™ cellular assays provide a proximal readout to analyze the phosphorylation status of STAT proteins in living cells. This system utilizes cell lines that stably express GFP-STAT fusion proteins. The phosphorylation state of GFP-STAT is then analyzed in cell lysates using a terbium (Tb)-labeled phospho-specific STAT antibody in a time-resolved, Föerster-resonance energy transfer (TR-FRET)–based readout. 

CellSensor® cell lines monitor the transcriptional function of the phosphorylated STATs using a beta-lactamase reporter gene assay in physiological and disease-relevant cell backgrounds. Beta-lactamase provides advantages over luciferase and beta-galactosidase reporter enzymes, in that it can be detected in living cells with its FRET-based membrane-permeable substrate. The dual-wavelength readout allows for ratiometric analysis, which significantly reduces experimental variables. 

Both LanthaScreen™ Cellular Assays (Figure 1) and CellSensor® Cell Lines (Figure 2) were validated by modulating the interferon/JAK/STAT pathway activation using both a small molecule inhibitor and a JAK-specific RNAi panel. The results suggest that these two assays can be used as integrated HTS-compatible tools for the analysis of JAK-proximal activity and JAK-mediated downstream pathway function in a physiological context.

LanthaScreen™ STAT1-U2OS Cellular Assay
Figure 1—Measurement of JAK-mediated STAT1 phosphorylation using the Lanthascreen™ STAT1 Cellular Assay. LanthaScreen™-STAT1 U2OS cells (K1469, 12,000 cells/well in 32 ml of assay medium, 384-well format) were plated the day prior to the assay. On the day of the assay, cells were pretreated with 4 ml of 1% DMSO before treatment with the indicated concentration of IFN-γ (4 ml addition) for 60 minutes. Cells were lysed by addition of 30 ml assay buffer (to 70 ml total volume) that included 5 nM Tb-anti-pSTAT1 (pY701) antibody (PV4844), and incubated for 60 min at room temperature. Fluorescence emission at 520 nm and 490 nm was obtained using a BMG Pherastar plate reader set to TR-FRET mode. The 520/490 nm emission ratios are plotted for each concentration of IFN-γ. {figure edits: remove title. y-axis: lower-case "r" in "ratio". x-axis: replace word "gamma" with gamma symbol; lower-case "l" in "ml".}
CellSensor® GAS-bla ME-180

Figure 2—Measurement of the transcriptional activity of STAT1 using the CellSensor® GAS-bla ME-180 reporter assay. GAS-bla ME180 cells (K1651, 20,000 cells/well) were plated in a 384-well plate and stimulated with IFN-γ over the indicated concentration range in the presence of 0.5% DMSO for 5 hours. Cells were then loaded with LiveBLAzer™-FRET B/G Substrate (K1095) for 2.5 hours. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and the emission ratios were plotted for the indicated concentrations of IFN-γ (n=16 for each data point). {figure edits: remove title. y-axis: space between numbers and units. x-axis: replace word "gamma" with gamma symbol; lower-case "l" in "ml".}

 


Interferons were among the first cytokines identified, and have since been characterized as having pleiotropic effects on the mammalian immune system that include antiviral and pathogen responses, inhibition of proliferation, cell differentiation, immune regulation, inhibition of angiogenesis, and apoptosis. Due to their role in antiviral and antitumor responses, interferons have been used as therapeutics in cancer and viral treatment.  Interferons are produced by multiple cell types in response to infection or challenge to the immune system that may include foreign antigens, viruses and parasites, or tumor cells. Interferons have been divided into several classes: Type I interferons (including IFNα, IFNβ) and Type II interferon (IFNγ). Both types signal through highly specific receptors, known as the IFN-α receptor complex (IFNAR1 and IFNAR2), and the IFNγ receptor complex (IFNGR1 and IFNGR2), respectively. 

Type I interferons have been shown to trigger apoptosis by activating the PI3K pathway, potentially through the phosphorylation of Akt and mTOR, which in turn activates downstream effectors such as 4E-BP1 and p70S6 kinase (S6K). While the Jak-STAT pathway is also triggered by IFNα, Jak-STAT and PI3K pathways are clearly distinct, as activation of the Jak-STAT pathway alone is not sufficient to trigger apoptosis in response to IFNα. Although mTOR is often considered to have anti-apoptotic effects, mTOR can phosphorylate p53 at serine-15, thereby triggering apoptosis through the mitochondrial cell death pathway, with activation of caspases 1, 2, 3, 8, and 9.

Among the best understood effects of interferons are their antiviral activities, one of the first activities ascribed to this class of cytokines. Antiviral activities of IFNα and IFNγ are primarily mediated through activation of the Jak-STAT pathway. Unlike the apoptotic effects of IFNα, antiviral activities are independent of PI3K/mTOR pathways; cells deficient in STAT1 are defective in IFN-mediated antiviral activity. While STAT1 is phosphorylated directly by IFNGR1 and JAK1/2, IFNγ activation also proceeds through a phosphorylation cascade that involves p38MAPK and PKCδ, leading to phosphorylation and dimerization of STAT1 and ultimately to transcriptional activation.

Although interferon signaling is mediated primarily through Jak-STAT phosphorylation, STAT-independent pathways have also been described, suggesting alternate means of IFN signaling and regulation of biological effects. Indeed, unlike IFNα signaling of apoptosis noted above, IFNb-dependent activation of STATs and p38 MAPK is not sufficient to fully inhibit proliferation of cells; rather, it works in cooperation with TSC2-dependent inhibition of PI3K/mTOR/S6K1 activity as demonstrated with human Lymphangioleiomyomatosis (LAM) and TSC2-null ELT3 cell proliferation (Goncharova et al., 2008). Moreover, it is apparent that there are other regulatory mechanisms that can finely tune activation and regulation of the Jak-STAT pathway itself. For example, STAT1 and STAT3, both components in IFN signaling, would appear to have opposite effects, as STAT1 activity is associated with tumor suppression, while STAT3 is considered an oncogene.

Monitoring the phosphorylation state of key participants in these various signaling cascades will undoubtedly lead to a better understanding of the various IFN signaling pathways, revealing more secrets to this complex and often puzzling paradigm.  Phosphorylation Site-Specific Antibodies (PSSAs) from Invitrogen are highly specific, robust antibodies which are validated on multiple platforms.

Detection of STAT3 [pY705] Phosphorylation (Dimerization/Nuclear Translocation Site) with Crude Lysates of 3T3 L1 AdipocytesFigure 1. Detection of STAT3 [pY705] Phosphorylation (Dimerization/Nuclear Translocation Site) with Crude Lysates of 3T3 L1 Adipocytes