Interferon Regulatory Factor 9 (IRF9) is a transcription factor that forms the ISGF3 complex with STAT1 and STAT2 to mediate type I interferon signaling. Upon interferon-α/β receptor activation, JAK1 and TYK2 phosphorylate STAT1 and STAT2, which then associate with IRF9 to form the active ISGF3 complex. This complex translocates to the nucleus and binds interferon-stimulated response elements (ISREs) to induce expression of interferon-stimulated genes (ISGs) involved in antiviral defense, immune activation, and cell cycle regulation. IRF9 is essential for establishing an antiviral state and coordinating innate immune responses. Dysregulation of IRF9 is implicated in autoimmune diseases, viral persistence, and cancer immune evasion. Therapeutic modulation of IRF9 activity holds promise for enhancing antiviral immunity and cancer immunotherapy.
The AlphaLISA SureFire Ultra Human Total IRF9 is a sandwich immunoassay for the quantitative detection of total IRF9 in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
Total-AlphaLISA SureFire Ultra two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Total-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.
IRF9 activation mediated by IFNβ
RPMI 8226 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and treated with 50 ng/mL of IFNβ at the indicated timepoints.
After treatment, the cells were washed with HBSS and then lysed with 100 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). IRF9 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 20,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
Treatment with IFNβ triggered a gradual increase to the Total levels of IRF9 with peak induction occurring 7 hours post treatment with no change to Total Cofilin levels (data not shown).
Type I and II interferons induce IRF9 in a dose-dependent manner
RPMI 8226 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and treated with increasing concentrations of IFNα, IFNb and IFNγ for 8 hours.
After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer or Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). Total IRF9 and Cofilin levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 20,000 cells for IRF9 and 400 cells for Cofilin) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, treatment with type I and type II interferons triggered a dose-dependent increase in the levels of Total IRF9 with no change to Cofilin Total.
Decrease of IRF9 levels by Ruxolitinib
THP-1 cells were seeded in a 96-well plate (300,000 cells/well) in complete medium and treated with increasing concentrations of Ruxolitinib for 24 hours.
After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). Total IRF9 and ERK1/2 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 30,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
Inhibition of JAK1 signaling mediated by Ruxolitinib, resulted in a significant decrease of Total IRF9 levels after 24 hours.
Knockout validation of IRF9 Total assay
Total IRF9 levels were assessed in HeLa wild type (WT) and HeLa IRF9 knockout (KO) (Abcam ab266051) cell lines. HeLa WT and KO cells were seeded in a 96-well plate (10,000 cells/well) and incubated overnight at 37°C, 5% CO2. Cells were treated with increasing concentrations of IFNα for 24 hours.
After treatment, cells were lysed in 100 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). IRF9 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of lysate (approximately 1,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
Total IRF9 was only detected in HeLa WT cells treated with IFNα, demonstrating assay specificity.
Total IRF9 expression in various cell lines
Adherent cell lines were seeded in a 96-well plate (40,000 cells/well) and incubated overnight at 37°C, 5% CO2. Cells were lysed with 100 µL of Lysis Buffer at RT with shaking (350 rpm).
Suspension cell lines were seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).
IRF9 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells and 40,000 suspension cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, basal Total IRF9 expression is low in most cell lines tested. Moderate levels of expression were detected in THP-1 cells.
