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.