Hey! I used mouse fibrosarcoma cell line (L929) and treated with 1ng/ml rmTNF-alpha and extracted protein at different time points (0min, 15min, 30min, 1hr, 2hr, 5hr, 8hr, 12hr). Then I performed western blotting to check the time-dependent profile of TNF-alpha signalling components. The p-p65 and p-p38 are showing less protein as compared to non-treated cells at 15min and 30min. 15min is showing relatively more protein than 30min. After 30min, p-p65 is showing increase in band strength while p-p38 is showing higher intensity 2hrs onward. Usually, there must be more phosphorylated form of these proteins but I am getting opposite profile. Is there any explanation for that? Should I change the time points? Did someone get the similar profile? Please guide me.
Your time points are ok. Several things to consider:
1. P-p65 and p-p38 signals in your non-treated cells should definitely be lower than your treated cells, regardless of the time point. If not, you have a problem.
2. P65 activation is relatively faster and more intense than MAPK. So if you see stronger signals early on for p65 (at 15 min compared to 30 min), it’s ok.
3. Not all signaling pathways are activated at the same rate. So p65 activation could be happening earlier than p38. Again, this is ok.
When you see phosphorylation of signaling proteins in your untreated cells, the addition of the stimulus seems to either not have an impact (no additional activation) or leads to a reduction in signals (which you seem to observe). Make sure your cells are not contaminated. If that’s not the problem, then try to rest your cells before addition of TNF, so your signaling pathways are in a basal state and can respond to activating signals appropriately.
Dear Nasir
Maybe you have to take inconsideration the translocation of this protein from cytoplasm to the nucleus
You can clarify that through checking of these proteins in both of cytoplasm and nucleus extractions
Another possibility is the degradation of the protein after phosphorylation
Best regards
Isehaq
Dear Ms. Julie and Mr. Isehaq,
Thank you for your valuable comments and suggestions. I also want to share one more result about IkBa degradation which was significantly observed as compared to non-treated control (no degradation at all). There was more degradation at 15min treatment time than 30min. After 30 min, no degradation was observed. This profile is in correlation with that of p-p65 except the higher p-p65 in control than treated one. It means that my treatment is degrading the IkBa (as expected) but not increasing the p-p65 (unlike expectation). Is it because of the degradation of the phosphorylated form (as Mr. Isehaq already suggested)? Can you please recommend any reference article to defend this result? Is it ok to publish only IkBa result to prove my concept?
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