I have an experiment that supposedly increases the JNK phosphorylation. However, the phosphorylation increased from basal levels at the p54 band, the p46 band on the other hand was on a plateau in a concentration-dependent manner. When the highest concentration (20uM) was co-incubated with the drug, the cells' viability decreased more. Could there be a difference in the function of the p54 and p46 bands?
By the look of things, you are getting a differential response for the two isoforms visible - the upper band increasing to maximal signal above control at the highest concentration, where the lower band rises from basal levels and then drops away again at the higher concentration. In answer to your question - yes, it is entirely possible that there is a difference in function between the two isoforms ; the question is what effect this has on downstream targets of the protein, where the isoforms are located, and how the drug interacts with the mechanism upstream of JNK phosphorylation.
Have you performed an actual cell viability assay to determine the effect of treatment - such as an ATP assay (ie., Cell Titer Glo G7572 from Promega) that could confirm that there is an impact on viability at the higher levels? What is the time course of this? The GAPDH band doesn't decrease at the highest levels suggesting that cell numbers / density are unaffected by the treatment, but this is obviously with the caveat that the time course may not be compatible with seeing an effect on cell death at this point.
@robin dickinson , the cells was collected after 12 hours. The highest concentration (20um) is the ld50 of the drug. I believe that high jnk would induce apoptosis of the cells but at lower concentrations that only induce low jnk will have protective mechanisms. When i added jnk inhibitor + 20um of the drug, it seems that it killed more cells compared to when drug was used alone. Thus, i think that at early stages, jnk has a protective mechanism.
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