Plenti6 v5 dest vector

The human AFAP1L1 gene was subcloned into a pLenti6/V5-DEST vector (Invitrogen, Carlsbad, CA). As a control, a β-galactosidase gene-expressing vector (pLenti6/V5-GW/LacZ, Invitrogen) was used. Stable cells were selected with blasticidin and several clones were isolated by limiting dilution. For the transient expression, the human AFAP1L1, AFAP1, and vinculin genes were subcloned into the pcDNA3.1+ plasmid tagged at the N-terminus with 3×Flag or 3×HA (Invitrogen). The transfection was carried out using Lipofectamine 2000 (Invitrogen).

The generation of plasmids allowing bacterial expression of GST-tagged full-length human USF2 and USF2 deletion fusion proteins in pGEX-5X-1 (GE Healthcare, Freiburg, Germany), as well as the plasmid pcDNA6A-Gal4-USF2, have been already described [28 (link)]. The expression vectors for wild-type (Addgene #1872) and kinase-dead (Addgene #1873) CDK5 were generous gifts from Sander van den Heuvel [57 (link)]. For the generation of the USF2 point mutants, the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Helsinki, Finland) was used. The pLenti6/V5-DEST™ vector (Invitrogen, Karlsruhe, Germany) has been used for the generation of lentivirus particles expressing non-phosphorylatable USF2-S155A/S222A and phosphomimicking USF2-S155D/T222D proteins.

The plasmids EpCAM-YFP, EpICD-YFP and YFP in the pEYFP-N1 vector backbone were a generous gift of Dr. Olivier Gires and are described by Maetzel et al. [17 (link)] The extracellular part of EpCAM with membrane anchor (EpEX-TM) was cloned by amplifying the fragment by the use of KOD polymerase (Novagen) and specific forward (5-TTA GTG AAC CGT CAG ATC CGC TAG C) and reverse primers (5-GGC GAC CGG TGA AAT AAC CAG CAC AAC). PCR fragments were digested with Nhe-I, Age-I (NEB) and ligated into the predigested p-EYFP-N1 vector (Nhe-I, Age-I, NEB) by the use of the Quick ligation Kit (NEB). Open reading frames were cut out by Nhe-I and Not-I (NEB), polished with Klenow (NEB) and ligated into the pENTR-11 Gateway vector (Invitrogen), predigested with Xmn-I and Eco RV (NEB) and polished with Klenow. All amplified and purified pENTR-11 vectors were sequenced for correct orientation and exclusion of incorporated mutations. pENTR-11 vectors were site-specifically recombined with the pLenti6-V5 DEST vector (Invitrogen) using the Gateway LR Clonase II Pus Enzyme Mix (Invitrogen). The resulting pLenti6 DEST vectors with the EpCAM-YFP, EpEX-YFP, EpICD-YFP and YFP open-reading frames (all amino acid sequences are provided in the Additional file 1: Figure S1) were transformed and propagated in One-Shot Stabl3 bacteria (Invitrogen).

The open reading frame of GPR3 cDNA (Genscript) and SIK2^{36 (link)} was cloned into a modified version of pLenti6/V5-DEST vector (Invitrogen) in which we deleted 300 nucleotides of the CMV promoter distal to the transcription start site were removed to attenuate its potency as per^{46 (link)}. The resulting expression plasmid, pLenti6-delta4/V5-DEST provides more physiological expression levels appropriate for genetic rescue experiments. shRNA-resistant constructs were generated by QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent). PCR oligo sequences and mutagenesis primer sequences are listed in Supplementary Data 4.