To check for protein degradation pathways how to make a solution of chemical inhibitors?

Dear Research Community,

I have recently started studying the MAPK-ERK Pathway in detail and using several inhibitors to block the signaling at different stages.

I have purchased the ERK1/2 inhibitor SCH7729 and treated a couple of lung adenocarcinoma cells for 48h, then harvested protein and performed Western Blot. And to my surprise, I could not see a reduction in phosphorylation of ERK1/2. Downstream targets of ERK1/2 were reduced though.

So I was wondering if anybody had similar experiences with this drug? It does seem to work with regard to target genes, but it does not inhibit phorphorylation of ERK1/2? I used a concentration of 10uM.

Are 48h treatment time too long?

Thank you for your feedback!

Is it possible to inhibit the TF expressed over the platelets by TFPI present over the endothelium?

Dear All,

When we transfected a gene into mammalian cells, we observed a particular protein disappeared in the cells. Therefore, we checked the expression of the particular protein in the presence of MG132, a proteasome inhibitor, and CQ & bafilomycin, autophagy inhibitors. Unfortunately, these inhibitors could not block the degradation of the protein. Is there any possible explanation for this phenomenon? (we tried calpain inhibitor as well)

Thanks in advance.

Are there specific inhibitors for either degradation pathway? From literature it seems that protease inhibitors like leupeptin also inhibit the proteasome?

I'd like to ask are there a reliable methods to find if protein degradation by proteosomal system is inhibited due to ER stress

Which pathway or signal should I check first ?

Hi, all. I want to test the Src kinase activity in my sample after a very short drug dosage. Does anyone know what kind of kit can we pick for a very quick detection?

Also, i am looking for a kind of specific inhibitor for c-Src kinase instead of PP2 which also works on other pathway like ABL or c-kit. Thanks!