I have been working on intermediate filaments modeling in brain cells. As i have knocked down a certain protein using siRNA, and i had performed SILAC based quantitative proteomics, results showed a significant increase in intermediate filament proteins. Upon validating this finding using western blotting and immunofluorescence, results showed a marked decrease in those proteins after treatment as well as showing almost diminished signal in immunofluorescence staining. I already looked into the isoforms detected by the mass spectrometer and compared it to the amino acid sequence that the antibody detects and found that the antibody detects a portion of the protein that is common between all isoforms for one of the intermediate filaments and for other intermediate filaments, there is only the one isoform. also i checked PTMs (except phosphorylation) for the peptides detected in the heavy versus light proteins and both were unmodified.

I am using the Fusion Lumos LC-MS/MS system.

I have been trying to find an explanation for this phenomenon and i would appreciate any help in this area.