Western blot
The cells were harvested in ice cold PBS, 48-72 hours after transfection. Lysate samples were prepared by homogenizing the harvested HEK293Tcells in lysate buffer (10mM Tris-HCl 6.8, 2.5% SDS, 2mM EDTA) containing protease inhibitor cocktail 2 (#P5726, Sigma) and 3 (#P0044, Sigma) and protease inhibitor (#P8340, Sigma). Protein concentrations were determined using the BCA kit (Pierce). Final working protein concentrations were adjusted to 1 mg/ml. Western blots were probed with primary antibodies against MAP3K7 (sc-7967, 1:1000, Santa Cruz), phospho-MAP3K7 (Thr187; #4536, 1:1000, Cel Signalling), ERK1/2 (#9102, 1:2000, Cell Signaling), phospho-ERK1/2 (#9101, 1:2000; Cell Signaling), Actin (MAB1501R, 1:20.000, Chemicon), RFP (#600401379, 1:2000, Rockland), NFKB (sc-514451, 1:1000, Santa Cruz), phospho-NFkb (sc-136548, 1:1000, Santa Cruz), GAPDH (2118S, 1:2000, Cell Signaling), TAB1 (67020-1-Ig, 1:10.000, Proteintech) and secondary antibodies (goat anti-mouse (#926-32210) and goat anti-rabbit (#926-68021), all 1:15.000, LI-COR). Blots were quantified using LI-COR Odyssey Scanner and Odyssey 3.0 software.