The overall goal of this procedure is to allow a detailed analysis of the phosphorylation-dependent activation of the IRF3 transcription factor. So this method can help answer key questions regarding the interferon mediate innate immune response. Such as a pathogen activates or invades the IRF3 dependent interferon beta expression.
The main advantage of this technique is that is constitutes affordable and sensitive approach to distinguish the different IRF3 activated form that may arise following different stimulations. Though this method provides insight into virus infection of human cells it can also be applied to mouse or human tissues subjected to other pathogens or pathogen-associated molecular patterns. Demonstrating the procedure will be Alexa Robitaille and Melissa Mariani, two cell Simulink experts from my laboratory.
The A549 cells used in this procedure are maintained in culture in 15 centimeter plate at 37 degrees Celsius and 5%carbon dioxide in complete F-12K Ham medium. Twenty four hours before infection, trypsinize the cells as described in the protocol text and transfer the cells to a 15 milliliter conical tube. Centrifuge at 350 x G for three minutes at room temperature.
Remove the supernatant and re-suspend the cell pellet in eight milliliters of complete F-12K Ham medium to obtain a homogeneous single-cell suspension. After counting the cells using a hemoctyometer, seed the cells at a density of one times tenth to the sixth cells per 60 millimeter plate, and four milliliters of preheated complete F-12K Ham medium. Incubate for 20 to 24 hours at 37 degrees Celsius, five percent carbon dioxide.
After 20 to 24 hours the cells should form a 90%confluent monolayer. Remove the medium and wash the cells with two milliliters of serum-free F-12K Ham medium, or SFM. Add two milliliters of fresh SFM per 60 millimeter plate.
Dilute to thawed aliquot of Sendai virus in preheated SFM to obtain 60 hemagglutination units, or HAU, per 100 microliters. Mix by pipetting up and down gently, and then add 100 microliters of diluted virus drop by drop to each plate to perform the infection at 40 HAU per 10 to the sixth cells. Do not add virus in the non-infected control plate.
Incubate the cells in the incubator at 37 degrees Celsius, five percent carbon dioxide. During the first hour of infection, agitate the plates by hand three or four times. At two hours post-infection add two milliliters of F-12K Ham medium containing 20%heat and activated FDS, to obtain a final concentration of 10%heat and activated FDS.
Incubate the cells in the incubator for one, four, and seven more hours to reach total infection times of three, six, and nine hours respectively. Cells will be harvested at each of these time points for preparation of whole cell extracts as demonstrated in the next video segment. Begin this procedure by removing the infection medium.
Harvest the cells by scraping in one milliliter of ice-cold DPBS. Transfer the cell suspension to a pre-chilled 1.5 milliliter centrifuge tube. Pellet the cells by centrifugation at 16, 000 x g at 4 degrees Celsius for 20 seconds.
And carefully decant all traces of DPBS. Re-suspend the cell pellet in 70 microliters of lysis buffer. Incubate on ice for 20 minutes.
Flash-freeze the lysate by incubation in a liquid nitrogen bath for 15 seconds. Then thaw the lysate at room temperature until it is completely melted. Vortex for ten seconds.
Repeat this freeze-thaw-vortex cycle three times. Centrifuge at 16, 000 x g at four degrees Celsius for 20 minutes. Transfer the supernatant, which is the whole cell extract, to a new pre-chilled 1.5 milliliter centrifuge tube.
It is important to keep the whole cell extracts on ice at all times. To resolve the whole cell extracts by high-resolution SDS-PAGE, prepare three denaturing electrophoresis gels, two 16 centimeter gels for the detection of IRF3 forms, and one 8.5 centimeter gel for the detection of Sendai virus proteins. Load 14 microliters of molecular weight standard in one well of each 16 centimeter gel.
Next load 30 micrograms of denatured whole cell extract per well of the two 16 centimeter gels. Load seven microliters of molecular weight standard in one well of the 8.5 centimeter gel. Load eight to ten micrograms of denatured whole cell extract per well of the 8.5 centimeter gel.
Run the gels at 30 milliamps constant current until the migration front reaches the bottom of the gel. Migration technically lasts approximately three hours for a 16 centimeter gel, and 45 minutes for an 8.5 centimeter gel. Begin this analysis by preparing a non-denaturing resolving gel of a minimum of 8.5 centimeters in length, following the procedure in the protocol text.
Press the running apparatus into ice approximately to the level of the lower chamber electrophoresis buffer, but make sure the gel is not in the ice. Pre-run the gel at 40 milliamps constant current for 30 minutes on ice. During the pre-run, mix the whole cell extracts kept on ice one-to-one with Two-X Native-PAGE loading buffer.
Immediately at the end of the pre-run, load eight to ten micrograms of whole cell extract on the gel. It is important to load the samples close to the bottom of the well so that the banding is not disturbed. Run the gel at 25 milliamps constant current on ice as demonstrated for the pre-run until the migration front reaches the bottom of the gel.
This will take approximately 40 minutes. At the completion of all gel runs, the proteins are transferred to nitro-cellulose membranes and fixed. The transfer procedure is described in the accompanying protocol text.
The three SDS-PAGE membranes, but not the Native-PAGE membrane, are stained with red Ponceau solution to identify the molecular weight markers. After incubating all four membranes from SDS-PAGE and Native-PAGE in blocking solution, incubate the membranes with the primary antibodies according to the sequential order shown in this diagram. Membranes must under go five five-minute washes in PBS-T before incubation with the appropriate horseradish peroxidase conjugated secondary antibodies.
After removing traces of tween with PBS, incubate the membranes for one minute in the volume of enhanced chemiluminescence reagent sufficient to fully cover the membranes. Use filter paper to dry the membranes and then place them in the luminescent image analyzer to visualize the immuno-reactive bands. Before incubation with the next primary antibodies, wash the membranes three times in PBS for five minutes each time.
When stripping is required between antibodies, incubate the membranes in pre-warmed stripping solution at 50 degrees Celsius for 20 minutes under regular agitation. Details of the immunoblot analysis can be found in the accompanying protocol text. Shown here is a typical immunoblot image of IRF3 detected with IRF3 total antibodies and IRF3 phosphospecific antibodies against Ser396 and Ser398, after high-resolution of whole cell extracts.
In unstimulated cells, IRF3 is detected as two bands corresponding to the nonphosphorylated, and the hypophosphorylated species of IRF3. Exposure of cells to Sendai virus results in a time-dependent shift to slowly migrating hyperphosphorylated forms three and four, which are also specifically immunodetected using the phosphospecific antibodies against Ser396 and Ser398. This next figure shows a profile of IRF3 detection obtained from whole cell extracts resolved by native PAGE, followed by immunoblot using anti-IRF3 NES antibodies, and phosphospecific antibodies against Ser386.
In unstimulated cells IRF3 is detected as a single band corresponding to the momomeric form. Upon infection with Sendai virus the levels of IRF3 monomer decrease with a concomitant accumulation of a slowly migrating band that corresponds to the dimeric form of IRF3, which is also specifically immunodetected using phosphospecific antibodies against Ser386. While attempting this procedure, it's important to remember that the composition and length of the different gels is critical to achieve an appropriate resolution of the IRF3 phosphoforms.
Following this procedure, other methods, like DNA binding or gene reporter acids can be performed in order to assess IRF3 activity. After watching this video you should have a good understanding of how to distinguish the different active forms of IRF3. Don't forget that working with pathogens may be hazardous and manipulations should be performed in accordance to the biosafety guidelines of your host institution.
