Figure 5

Complexes of mOSM-RFPs and mOSM analyzed by bn-PAGE. (A) 35 ng of mOSM were incubated with 200 ng of the respective mOSM-RFP (with the exception of i-mOSM-RFP that is less efficiently expressed and therefore 200 ng could not be achieved) in 50 μl for 30 min. Subsequently Coomassie Brilliant blue G-250 was added and the protein complexes were separated on a native gradient gel (4-16% PAA). After blotting of the proteins to a PVDF membrane mOSM-RFPs were detected using a FLAG antibody (upper panel). After stripping of the blot mOSM was detected using a mOSM antibody (middle panel). (B) bn-PAGE was performed as described in (A) with the protein amounts indicated in the figure. After blotting, detection of the proteins was performed with primary goat-anti-mOSM and mouse-anti-flag antibodies followed by secondary rabbit-anti-goat-Cy2 and donkey-anti-mouse-Cy3 antibodies. Fluorescence was detected with a fluorescence scanner. Afterwards the mOSM antibody was visualized by ECL using a matching HRP-conjugated secondary antibody.