We are currently conducting gene function studies using overexpression and knockdown approaches. We have observed that some transduced genes resulted in decreased proliferation of fibroblast cells. We want to study potential pathways involved in this observation such as Akt, Jak/Stat pathways including their phosphorylation status. After seeding cells, when should we collect cells to study these pathways using western blot (eg. 24h, 48h, 72h or the fastest dividing cells reached %80 confluence)?
Thanks in advance
Hi, Selami, I am not sure one can tell you exactly correct incubation time. Actually, you have use same time for all tretaments, much better if you study kinetcs of cell proliferation with parallel investigation of gene exoression. You can try to use EdU as nirmalization marker. Probably, you need to use at least 3-4 different time points for EdU and WB and establish a causla link between your gene of interest abd cell proliferatioon.
Best wishes!
- Badges
- Science method