In some articles a colocalization is found between microglial marker OX-42 and phosphorylated p38 MAPK. However the antibody I used (cell-signalling) only labels nuclear P-p38 and therefore I see no colocalization with OX-42. All of the antibodies from cell-signalling label nuclear P-p38 as far as I know, however there are articles describing colocalization with these antibodies as well. How to explain this?
Dear Maarten,
maybe you have to better interpret what the authors exactly indicate with "colocalization". It is well known that, once activated, a great part of P-p38 translocates into the nucleus and phosphorylates several substrates, thus it is usual that you observe a strong nuclear localization of this protein. However, it is likely that the colocalization you mention is not a SUBCELLULAR colocalization, but a colocalization with OX-42 in terms of cell types. Probably, the articles you read reported an increase in p38 activation in microglia (since the cells positive for P-p38 were also positive for the microglial marker OX-42) not seen in other cell types (astrocytes, oligodentdrocytes, neurons).
I hope i was clear in my explanation...however, this is just my personal interpretation!
p38 MAPK is a signaling molecule, hence its localization in cell is not fixed and highly dependent on environmental condition in which cell is present. Depending on presence stimulator or inhibitor this molecule may be present in cytoplasm or in nucleus. It actually shuttle between nucleus and cytoplasm depending its state of phosphorylation
Dear Maarten,
maybe you have to better interpret what the authors exactly indicate with "colocalization". It is well known that, once activated, a great part of P-p38 translocates into the nucleus and phosphorylates several substrates, thus it is usual that you observe a strong nuclear localization of this protein. However, it is likely that the colocalization you mention is not a SUBCELLULAR colocalization, but a colocalization with OX-42 in terms of cell types. Probably, the articles you read reported an increase in p38 activation in microglia (since the cells positive for P-p38 were also positive for the microglial marker OX-42) not seen in other cell types (astrocytes, oligodentdrocytes, neurons).
I hope i was clear in my explanation...however, this is just my personal interpretation!
I agree both with Ravinder and Marco that p38 Mapk is a shuttle signaling molecule travelling from cytoplasm to the nucleus that may also localize to mitochondria and autophagosomes in certain disease states "dementia" (Zhu et al., 2003). Thus, it may be considered an inducible kinase whose exact localization is also dependent on the stimuli that cause its activation. In your case colocalization with Ox-42 might depend on the fact that microglial cells are activated by some stimulus that transiently causes activation of Mapks. If this is the case try to perform detections at different time points.
I agree with all of you about the function and translocation of P-p38 to the nucleus. However I would like to stress that I was not talking about a colocalization in terms of cell types. There are articles (Ru Rong Ji et al. and C. Svensson et al. for example) that show colocalization in terms of green + red = yellow.
I only find P-p38 in the nucleus. Not one microglial cell shows double labeling (as in yellow) while I stain for the same form of the protein (same company, just a newer version of the antibody). If it would be dependent on the state of p38 (phosphorylated or not) you would always expect one form in the cytoplasm and the other in the nucleus right? They found the phosphorylated form colocalizing with OX-42 while I find none at all.
Even if this depends on other influencing factors I would expect the localization to be different to some degree but not in total right? I would expect at least some P-p38 to colocalize with OX-42 regarding the fact others find it.
I hope someone can clarify this to me. Thanks
i completely agree with Alessandro. One can perform time dependence study of localization in control and stimulated cells along with other established marker which may help in knowing proper localization i.e. whether it is in nucleus, cytoplasm or i n other cellular compartment.
Dear Maarten,
We have performed phospho p38 in eye lens tissues of control and some gene knockout mice. We observed clear nuclear staining as well as visible cytoplasmic staining. Since the level of cytoplasmic phospho p38 is expected to be low, fresh tissue sectioning /cell preparation is essential to detect its low levels in cytoplasm.
totally agree. Look these signal molecules remain in cell mostly in cytoplasm. Only when cell receive signal than the level of these molecules changes in cytoplasm as activated molecules moves in nucleus. Even in stimulated condition on expect low signal in cytoplasm and higher in nucleus. Although after some time it is possible that signal intensity becomes equal in both cytoplasm and in nucleus and further at later stage higher signal in cytoplasm and lesser or no signal in nucleus
Since I just got interested in staining p38-MAPK, I searched in the literature and found in one article that the two p38 isoforms alpha and beta localize differently: Lee et al., J Neurosci Res, 2000. Constitutive Activity and Differential Localization of p38a and p38b MAPKs in Adult Mouse Brain. They said that "Especially in hippocampus, p38alpha was heavily expressed in dendrites as well as in cell bodies of pyramidal neurons in CA1 and CA3 regions, and p38beta was preferentially in nucleus of neurons". So maybe there is a discrepancy in localization also of the activated forms.
- Badges
- Science method