Lentiviral Vectors for TTL Expression

For lentiviral experiments, vector eGFP-pWPT (Addgene no. 12255, kind gift from D. Trono) was used to express eGFP, and cDNA encoding human TTL (NP_714923, Origene no. RC207805L2) was cloned in it for TTL expression. PCR amplification and cloning of TTL cDNA were performed with Phusion DNA polymerase (Thermo Scientific) and In-Fusion HD Cloning kit (Clontech), respectively. eGFP cDNA was removed during the cloning process to produce an untagged TTL. For lentiviral shRNA expression, two TTL shRNA sequences, cloned in pLKO.1 vector, were purchased from Sigma-Aldrich: shTTL1 (TRCN0000191515, sequence: 5′-CCG GCA TTC AGA AA GAG TAC TCA ACT CGA GTT GAC TAC TCT TTC TGA ATG CTT TTT TG-3′) and shTTL2 (TRCN0000191227, sequence: 5′-CCG GCT CAA AGA ACT ATG GGA AAT ACT CGA GTA TTT CCC ATA GTT CTT TGA GTT TTT TG-3′).³⁵ The SHC001 pLKO.1-puro empty Vector (Sigma) was used as control (shControl). For the transfection experiments, the plasmid encoding pCMV-EB3-EGFP was a kind gift from Dr Frank Polleux.^{72 (link)} Kind gifts from Dr Erik Dent include the plasmids EB3-tdTomato (Addgene no. 50708) and the plasmid encoding DsRed2 (Clontech), cloned into a pCAX vector. The plasmid pEGFP-N1 with a CMV promoter was also used (Addgene no. 6085-1). All constructs were verified by sequencing (Eurofins and Genewiz). Plasmids were purified with HiPure Plasmid Maxiprep kits (Invitrogen).