Maybe via the phosphorylation pattern of down stream effectors (e.g. see J Am Coll Cardiol. 2013 Nov 5;62(19):1737-45 or Environ Sci Pollut Res Int. 2020 Oct 30. doi: 10.1007/s11356-020-11416-5).

Thank you so much Andreas Eisenreich for these references.

In short, if you want to examine TLR receptors activation by your interest of protein, you can directly do either commonly used methods like Western Blot or Histology on certain TLR receptor activation (TLR2, TLR4 etc.). This is for the upstream activation check. For downstream signaling activation check, you can do pStat with WB method, which is the simplest efficient way, and can also do ELISA or qPCR for cytokines activation/expression.

From Invivogen, there is HEK blue cell line transfected with Il-10 receptors and it was mainly designed to activate TLRs pathway via Jak STAT mechanism for biochemical assay. These cells can also be used for screening anti-IL-10/10R antibodies.You can use this cell line for TLR and JAK/STAT signalling. https://www.invivogen.com/hek-blue-il10

Hello Hassan, our functional assay might also be usefull, in which we can measure kinase activity within your lysate differentially between samples. And because it contains many different kinase targets, you can investigate the whole pathway, up and downstream at once. Please read more about it at https://pamgene.com/science/

Or mail me directly if you have further questions [email protected]

Dear Hassan Abdulrazzak

It is quite possible with flowcytometry. If you want to check signalling via these molecule you must treat your cells with the specific stimulant followed by fixation and permeablization and surface/intracellular staining. The sample can be acquired on flowcytometry and it will give you relative results (upregulation/downregulation in comparision with controls).

If you want to perform flowcytometric assays for these signalling, let me know. I will send you references.

All the best.

Jitendra