To generate the FLAG-MITF-A expression vector, a FLAG sequence was inserted at the SmaI and HindIII restriction sites.
To generate the FLAG-tagged SF1, the full-length open reading frame lacking the stop codon was amplified by PCR and then subcloned into pCAGGS-FLAG (Hasegawa et al. 2010).
To generate the Flag-tagged HER3ΔNLS2 construct, the coding sequence was amplified by PCR (primers used were: 5′-GGGGTACCGAGGGCGAACGACGCTCTG-3′and 5′-GCTCTAGATTACGTTCTCTGGGCATTAGC-3′) and subcloned into the Kpn I and Xba I sites on the pFlag-CMV3 vector (Sigma-Aldrich, St Louis, MO, USA).
To generate the pUAST-Flag-RING-PR, the following two poly-nucleotides PR-F 5'-aattcacctcccctgccaccccgat-3' and PR-R 5'-ctagatcggggtggcaggggaggtg-3' were synthesized in vitro, and they were ligated to the pUAST-Flag-RING.
To generate the N-terminal Flag-tagged Sac1 (Flag-Sac1) construct, the full-length Sac1 cDNA was amplified from clone GH08349 with PCR primers containing BamHI and SalI sites and cloned in-frame into the pCMV-Tag2b vector (Clontech).
The resulting plasmid, pCMV-Tag4A-3X-Flag, conthens the insertion DYKDHDGDYKDHDI in front of the existing Flag-tag DYKDDDDK, thereby generating the triple Flag epitope.
The Flag-TDP-43 product was blunted and then cloned in pCDNA5FRT/TO vector previously digested with PmeI enzyme to generate the construct pCDNA5FRT/TO-Flag-TDP-43.
(A ) Schematic of the domain structure of the full-length mouse Chd1 (1-1711) used to generate the N-terminal FLAG-tagged construct.
cDNAs encoding rat USP2a (AF202453), USP2b (AF202454) or their shared core region (Lin et al., 2000; Lin et al., 2001) were subcloned into the BamHI and XhoI sites of the modified pRK5 to generate the C-terminal Flag- or HA-tagged USP2a, 2b, and USP2 core proteins, respectively.
The vector pcDNA3.1-FLAG-CBP was used to generate the deletion series pcDNA3.1-FLAG-CBP 1 507, 1 1100, 1 1458 and 1 1901 by digestion with BlnI, XbaI, XhoI and XmaI restriction endonucleases, respectively, followed by gel purification and re-ligation of the appropriate fragments.
