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.