Hi, I have been working on Her2 stimulation and doing western blot on P-her2 inhibition with our synthesized anti her2 and EGFR inhibitors . I used NRG(HRG) to stimulate the phosphorylation of her2 in different cell lines which I was working with. p-HER2 Ab. is from cell signaling but it seems is not as specific as expected and the stimulation is not really as clear as it is with EGF stimulation of EGFR. Any help will be appreciated in advance.
Dear Nahid,
Her2 receptor differs from other ErbB proteins by its structure; it is able to dimerize without cognate ligands. Therefore, Her2 activation by ligands depends on hetero-dimerization with other ErbB members. Neuregilnns (NRG, HRG) are ligands for ErbB3 and ErbB4, and not for Her2. So, you should be sure that at least one of this receptors is functionally expressed in your cell lines. It is important to correctly design your experiment! In particular, ErbB4 is not expressed in many cancer cells, such as breast, lung, etc. If you cell line harbours ErbB4 gene, I suggest you making control experiment by checking its phosphorylation with an antibody specific for its phoshprylation site(s). From another side, ErbB3 lacks of kinase domain, but the ligand binding to this receptor activates Her2 phosphorylation through heterodimerization.
I do not think that the problem is related with anti-pHer2 AB from CST that is known by good quality of anti-phosphorylated antibodies. By the way, this company sells the anti-pTyr antibody (including biotinylated version) that recognizes "pan tyrosine epitope". It might be useful for you in rapid checking the ability of neuregulin(s) to stimulate phosphorylation in your cell lines. If it works, you could study the activation of Her2 by antibodies specific for phosphorylation of particular amino acids.
Best wishes
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