Constructs
The MAP3K7WT (NM_003188.4) cDNA was obtained
from the human brain library by PCR (Phusion high fidelity, Thermo
Fisher) using the following primers: FW 5’-GTCTCACCCGGATTGTCC; RV
5’-AATGTAACGGTCCCAGAG. To be able to clone this sequence into our dual
promotor expression vector (Proietti Onori et al., 2018), the fragment
was tagged with restriction enzymes AscI; PacI by PCR (Phusion high
fidelity, Thermo Fisher) using primers: FW 5’-
GAATTGCGGCGCGCCACCATGTCTACAGCCTCTGCC; RV 5’-
GAATCCTTAATTAATCATGAAGTGCCTTGTCG. The point mutations were introduced
with site-directed mutagenesis (Phusion high fidelity, Thermo Fisher)
using the following primers: MAP3K7 c.143G>A
(p.Gly48Glu), FW 5’-GGAAGAGGAGCCTTTGAAGTTGTTTGCAAAGCT and RV
5’-AGCTTTGCAAACAACTTCAAAGGCTCCTCTTCC; MAP3K7 c.
208G>C (p.Glu70Gln), FW 5’-
TAAACAAATAGAAAGTGAATCTCAGAGGAAAGCGTTTATTGTAGA and RV 5’-
TCTACAATAAACGCTTTCCTCTGAGATTCACTTTCTATTTGTTTA; MAP3K7c.248G>A (p.Arg83His), FW 5’-
GCTTCGGCAGTTATCCCATGTGAACCATCCTAATA and RV 5’-
TATTAGGATGGTTCACATGGGATAACTGCCGAAGC; MAP3K7 c.
299T>A (p.Val100Glu), FW 5’-
GGAGCCTGCTTGAATCCAGAGTGTCTTGTGATGGAATAT and RV 5’-
ATATTCCATCACAAGACACTCTGGATTCAAGCAGGCTCC; MAP3K7c.329G>A (p.Gly110Asp), FW 5’-
GATGGAATATGCTGAAGGGGACTCTTTATATAATGTGCTGC and RV
5’-GCAGCACATTATATAAAGAGTCCCCTTCAGCATATTCCATC; MAP3K7c.337T>G (p.Tyr113Asp), FW
5’-TGAAGGGGGCTCTTTAGATAATGTGCTGCATGG and RV
5-CCATGCAGCACATTATCTAAAGAGCCCCCTTCA; MAP3K7 c.502G>C
(p.Gly168Arg), FW 5’- CTTACTGCTGGTTGCAGGGCGGACAGTTCTAAAAATTTG and RV 5’-
CAAATTTTTAGAACTGTCCGCCCTGCAACCAGCAGTAAG; MAP3K7c.586A>G (p.Met196Val), FW 5’-
GGGAGTGCTGCTTGGGTGGCACCTGAAGTTT and RV
5’-AAACTTCAGGTGCCACCCAAGCAGCACTCCC; MAP3K7 c.617A>G
(p.Tyr206Cys), FW 5’- GAAGTTTTTGAAGGTAGTAATTGCAGTGAAAAATGTGACGTCTTC and
RV 5’- GAAGACGTCACATTTTTCACTGCAATTACTACCTTCAAAAACTTC; MAP3K7c.616T>G (p.Tyr206Asp), FW 5’-
GTTTTTGAAGGTAGTAATGACAGTGAAAAATGTGACG and RV 5’-
CGTCACATTTTTCACTGTCATTACTACCTTCAAAAAC; MAP3K7c.721T>G (p.Trp241Gly), FW 5’-
CCCAGCTTTCCGAATCATGGGGGCTGTTCATAATGGTAC and RV 5’-
GTACCATTATGAACAGCCCCCATGATTCGGAAAGCTGGG; MAP3K7c.1454C>T (p.Pro485Leu), FW
5’-ACTACAGCCTCTAGCACTGTGCCCAAACTCCAAAG and RV
5’-CTTTGGAGTTTGGGCACAGTGCTAGAGGCTGTAGT. For the in vivoexperiments the ‘empty vector’ serves as control, consisting of the dual
promotor expression vector only (no gene insertion in MCS).
The TAB1wt (NM_006116.3) cDNA was obtained
from human brain library by PCR (Phusion high fidelity, Thermo Fisher)
using the following primers: FW 5’-ATGGCGGCGCAGAGGAG and RV 5’-
CTACGGTGCTGTCACCAC. This was then also cloned in our dual promotor
expression vector, tagging the fragment with restriction enzymes AscI;
PacI by PCR (Phusion high fidelity, Thermo Fisher) using primers: FW
5’-GGCGCGCCACCATGGCGGCGCAGAGGAG and RV 5’-TTAATTAACTACGGTGCTGTCACCAC.