HEK293T transfection
To assess the functional effects of the different MAP3K7variants, HEK293T cells were cultured in DMEM/10% Fetal Calf Serum
(FCS)/1% penicillin/streptomycin in 6-wells plates and transfected with
the following constructs: Control vector;MAP3K7WT; MAP3K7G48E;
MAP3K7E70Q; MAP3K7R83H;
MAP3K7V100E; MAP3K7G110D;
MAP3K7Y113D; MAP3K7G168R;
MAP3K7M196V; MAP3K7Y206C;
MAP3K7Y206D; MAP3K7W241G;
MAP3K7P485L; TAB1WT. In single
construct transfections, 3ug construct per well was transfected. When in
co-transfection of the different MAP3K7 constructs withTAB1WT , 1.5 µg/construct/well was transfected.
Constructs for co-transfection were mixed before actual transfection,
allowing both constructs to be transfected at the same time. At
subconfluency (60-70%), cells were transfected with PEI according to
the manufacturer instructions (Sigma). To avoid toxicity of prolonged
exposure to PEI, the medium was refreshed after 4-6 hours. Transfected
cells were used to prepare protein lysates for Western blot.