This protocol utilizes a widely-used technique along with reagents and tools that can be acquired commercially to assess endogenous IRF5 activation during the early events of stimulation. It can easily be applied for probing IRF5 biology in other cellular context. While native PAGE is a widely-used technique, obtaining clear, robust, interpretable, and reproducible results can be technically challenging.
Using a commercial running buffer and gel system helps to minimize variability. And paying attention to details is essential, and experience is key to success in obtaining good results. This modified native PAGE protocol involves many steps with subtle details that are key to success.
Visual demonstration can show these details that would be beneficial for researchers. Maintain CAL-1 cell culture in a T75 flask at 37 degrees Celsius and 5%carbon dioxide, with complete RPMI 1640 medium. When ready to stimulate the cells, transfer them to a 50-milliliter conical tube and centrifuge them at 200 x g for five minutes.
Remove the supernatant, and re-suspend the pellet in medium to obtain a homogeneous single cell suspension. Then account the cells with a hemocytometer, and seed them at a density of 1, 000, 000 cells per well in a six-well plate with four milliliters of pre-heated medium in each well. Incubate the cells for 20 to 24 hours to allow the confluency to reach 90 to 95%The next day, stimulate the cells by adding four microliters of one milligram per milliliter R848 per well, making sure to leave a control well with cells and no R848 treatment.
Gently rock the plate from side to side to evenly disperse the R848, then incubate the cells for 2 to 16 hours at 37 degrees Celsius and 5%carbon dioxide. After the incubation, transfer the cell suspensions from the plate into 5-milliliter centrifuge tubes. Centrifuge the cells at 200 x g for five minutes, then remove the supernatant, and re-suspend the pellet in one milliliter of PBS.
Transfer the cell suspension into a 1.5-milliliter tube, and spin it down at 12, 000 x g for 30 to 60 seconds at 4 degrees Celsius, then carefully remove the supernatant. Prepare the lysis buffer according to the manuscript directions, and keep it on ice until ready to use. Re-suspend the cell pellet in 30-microliters of ice-cold lysis buffer, and pipette up and down to mix.
Then incubate the tube on ice for 15 to 20 minutes. Clarify the lysate by centrifuging at 12, 000 x g for 15 to 20 minutes at 4 degrees Celsius, and transfer the supernatant to a pre-chilled 1.5-milliliter tube, making sure to keep the extracts on ice at all times. Then, measure the protein concentration using Bradford Reagent.
Prepare the upper-and lower-chamber electrophoresis buffers according to the manuscript directions, and rinse a 3-12%native PAGE gel thoroughly with water, without distorting the wells Extra care should be taken when handling the sodium deoxycholate. PPE for protection such as lab coat, safety goggles, and mask are required for handling the chemical. Then, set the gel into the Mini Gel Tank, remove the comb, add the prepared upper-and lower-chamber electrophoresis buffers, and pre-run it in a 4 degrees Celsius cold room or on ice at 150 volts for 30 minutes.
Meanwhile, prepare the samples for loading by mixing the cellular proteins on ice with 4X Native Sample Buffer. Once the pre-run is complete, load 10 to 15 micrograms of protein to each well, and run the gel for 30 minutes at 85 volts, followed by two hours at 150 volts. Then, soak the gel in SDS running buffer for 30 minutes at room temperature.
Activate the polyvinylidene difluoride membrane by soaking it in methanol for approximately five minutes. Make a cut on one corner of the membrane to indicate its orientation, and assemble the transfer sandwich according to the manufacturer's protocol. Place the transfer cassette into the tank and transfer at 20 volts for one hour on ice.
Then remove the membrane from the cassette with plastic forceps and block the membrane in blocking buffer at room temperature for 45 minutes on a rocking shaker. Next, incubate the membrane with the primary antibody at 4 degrees Celsius overnight or at room temperature for two hours and wash it with 1XTBST washing buffer for three minutes while rocking. Then incubate the membrane with the secondary antibody again using the 1XTBST washing buffer at room temperature for 45 minutes, repeating the washes.
Then scan the blot using an appropriate gel documentation system. Using this protocol, CAL-1 cells that were either stimulated or unstimulated with R848 were analyzed with an immunoblot. In unstimulated CAL-1 cells, IRF5 was detected as a single band on the native PAGE corresponding to its monomeric form.
For the stimulated cells, the level of IRF5 monomer decreased, while the level of the dimer increased. An immunoblot with anti-IRF5 antibody was performed on IRF5 over-expressing 293 T cells transfected with various constructs. No IRF5 was detected in the untransfected control, demonstrating the specificity of the anti-IRF5 antibody.
A single band corresponding to monomeric IRF5 was only detected in the 293 T cells over-expressing IRF5. When constructs encoding IRF5 activating proteins were co-transfected, a slowly-migrating band corresponding to the dimeric form of IRF5 appeared. However, NMDA5, a related protein to RIG-I, did not induce IRF5 dimerization.
The use of Bis-Tris gradient gels is crucial, likely due to the specific pH and chemical composition of this gel electrophoretic systems that allowed separation of monomeric and dimeric forms of IRF5. Also, lysing and preserving cell lysates in non-denaturing native sample buffer retains native protein structures. Here we used a commercially-available one that is tailored for native PAGE acids.
An additional method that compliments greatly with this is, is to utilize the ImageStream imaging flow cytometry system to assess IRF5 nuclear translocation, which is the substance that in IRF5 activation after dimerization. When combined with our protocol, it serves as an other kind of test to validate the steps involved in IRF5 activation. Being a key regulator of the inflammatory response, IRF5 plays important role in infection and immunity, autoimmune diseases, cancer, and many other diseases important for human health.
There are also efforts in developing therapeutics to target IRF5 and related transcription factors. This protocol will allow researchers in diverse fields to understand and probe IRF5 biology.
