Detecting Protein Interactions Using Tags

MEFs^Keap1−/− cells were lysed as previously described [68 (link)]. Cell lysates were separated by SDS-PAGE, and proteins were blotted to PVDF membranes. For detection, we used antibodies against the tags of the transfected proteins of interest in order to avoid the interference of endogenous proteins and the poor performance of commercially available antibodies for our purpose. Antibodies were used against Myc-Tag (#2276S, 1:1000 dilution) from Cell Signaling (Danvers, MA, USA), HA-Tag (#3724S, 1:1000 dilution) from Cell Signaling, beta-actin (#8457S, 1:1000 dilution) from Cell Signaling and FBXW11 (#13149-1-AP, 1:000 dilution) from Proteintech (Rosemont, IL, USA). Densitometric quantification was performed with ImageQuant™ TL, Cytiva (Tokyo, Japan)