The overall goal of this experiment is to identify the downstream signaling cascade involved in cells stimulated with mitochondrial-derived peptides. This method can answer key questions in the mitochondrial-derived peptide field such as what signaling pathways are involved, and where this occurs. The main advantage of this technique is that it is straight forward and relatively easy to perform.
We first had the idea for this method when we examined humanin-mediated ERK signaling which gave us limited information on the downstream facts. The Subscale Kersher method gave us a better idea of the role of humanin-mediated signaling. On the third day after plating and washing two times ten to the sixth, SH-SY5Y or HEK293 cells according to the text protocol.
Dissolve S14G humanin peptides in zero point two micron filtered distilled water. And re-constitute them as a one millimolar stock solution. Aliquot the pre-warmed serum-containing or serum-free medium in a 15 milliliter conical tube and add room temperature humanin peptide stock solution to make a working concentration.
Immediately replace the medium in the cells with six milliliters of the peptide solution. And incubate the cells for the appropriate amount of time. Use 10 milliliters of ice-cold PBS to wash the cells twice.
Then scrape the cells off the plate in five milliliters of ice-cold PBS. And transfer the cell suspension into a 15 milliliter conical tube. Centrifuge the cells at 500 times G and four degrees Celsius for five minutes.
Re-suspend the pellet in 200 microliters of ice-cold fractionation buffer and incubate on ice for 15 minutes. Centrifuge the tube at 250 times G and four degrees Celsius for five minutes. Then collect the supernatant as the cytoplasmic fraction and keep the pellet for the nuclear fraction.
Centrifuge the supernatant to remove cellular debris and other contaminants at 18000 times G and four degrees celsius for ten minutes. And then transfer the supernatant into a new micro-centrifuge tube. This is the cytisol fraction.
Re-suspend the pellet in 200 microliters of ice-cold wash buffer and centrifuge the solution at 250 times G and four degrees Celsius for five minutes. Remove the supernatant and use 100 microliters of ice-cold nuclear extraction buffer to re-suspend the pellet. Then sonicate the sample on ice 10 times.
Centrifuge the sample again. Then transfer the supernatant to a new microcentrifuge tube. To isolate the crude mitochondrial fraction, use 10 milliliters of ice-cold PBS to wash the cells in each 10 centimeter dish.
Then add five milliliters of ice-cold PBS and use a cell-scraper to detach the cells. Transfer the cell-suspensions into a single 15 milliliter conical tube. Then centrifuge the cells at 600 times G and four degrees Celsius for 10 minutes.
Aspirate the PBS and use one milliliter of ice-cold mitochondrial isolation buffer to re-suspend the pellet. Then with a polytetrafluoroethylene-coated pestle on ice, homogenize the cells with 25 strokes of a two milliliter homogenizer. Transfer the homogenate to a micro-centrifuge tube and centrifuge the sample at 600 times G and four degrees Celsius for 10 minutes to remove the nuclei and unbroken cells.
Then collect the supernatant, transfer it to a new micro-centrifuge tube and centrifuge the solution at 7000 times G and four degrees Celsius for 10 minutes. After removing the supernatant, re-suspend the pellet with 200 microliters of ice-cold mitochondria isolation buffer and transfer the solution to a new micro-centrifuge tube. Centrifuge the tube at 7000 times G and four degrees Celsius for 10 minutes.
After repeating the wash, use 15 microliters of RIPA buffer to re-suspend the pellet and incubate the suspension on ice for 10 minutes. Then centrifuge the sample at 16000 times G and four degrees Celsius for 15 minutes. Transfer the supernatant, which is the mitochondrial fraction, into a new tube.
Quantify and carry out western blotting according to the text protocol. Using the procedure demonstrated in this video, HEK293 and SH-SY5Y cells retreated with one and 100 micromolar S14 G humanin, a potent humanin analog. And the total and phosphorylated form of ERK12 at theanine 202, tyrosine 204 from total protein extracts were analyzed.
As seen here, S14G humanin treatment showed an increase in phosphorylation of ERK12 and its regulatory theanine-202, tyrosine-204 site. To understand the target of S14G humanin mediated ERK12 activation, the sub-cellular localization of total and phosphorylated ERK12 following S14G humanin treatment, was analyzed. As shown in these western blots, in HEK293 cells, the phosphorylated ERK increased in both the cytoplasm and nucleus but not in the mitochondria.
In SH-SY5Y cells however, phosphorylated ERK decreased in the cytoplasm nucleus and mitochondria. Suggesting that S14G humanin-mediated ERK phosphorylation may play a different role in different cell types and different conditions. Once mastered, the cell nuclear fractionation can be done in one hour and the mitochondrial fractionation can be done in one hour and 30 minutes, if it is performed properly.
While attempting this procedure, it is important to remember to keep all samples on ice. Following this procedure, other methods including co-immunal precipitation and mass spectrometry can be performed in order to understand the downstream targets of the signaling molecules.
