Hello everyone, I am trying to detect pIRF3 protein in MDA-MB-231 and BT-549 cells, but have been unsuccessful for a while now. I am able to detect other proteins such as pSTAT1 and pSTAT3, but pIRF3 just doesn't seem to work even after stimulation of the cells. Has anyone tried to detect this protein in these cell lines? If so, what are the protocols and antibodies used?

My protocol:

- After transferring proteins onto nitrocellulose membrane, block in 5% milk/ TBST for 45mins

- Incubate with primary antibody in 5% milk/TBST or 5% BSA overnight (Antibody from Cell Sig Tech #4947s, 1:500)

- Incubate with anti-rabbit antibody (1:2000) in 5% milk/TBST for 2h before proceeding with detection using Super signal West Femto

Thank you.