Pegfp plasmid

Using first strand cDNA obtained from a healthy donor as templates, the full-length coding sequence of RRP7A (GenBank accession number NM_015703.4) was PCR amplified and cloned into the mammalian expression vector pEGFP-n1 using EcoRI and BamHI restriction sites. Two full-length wild-type expression constructs for RRP7A were generated; one containing a green fluorescent protein (GFP)-tag at the C-terminus and another containing a FLAG-tag at the N-terminus and a C-terminal GFP-tag, using pEGFP plasmid (Addgene). The mutation p.W155C was introduced into these vectors using QuikChange II Site-Directed Mutagenesis Kit (Agilent technologies Inc.). The primers used were 5′-GGCATTCACAAGTGCATCAGTGACTACGC-3′ (sense) and 5′-GCGTAGTCACTGATGCACTTGTGAATGCC-3′ (anti-sense). Expression constructs were verified by Sanger sequencing, and expressed in RPE-1 and HEK293T cells. Cells were seeded in DMEM without pen/strep, and transfected using FuGENE6 HD Transfection Reagent (Promega) when they reached ~60% confluence^{69 (link)}. As a control in IP analyses, HEK293T cells were also transfected with the empty vector pEGFP-C1.

Transfections of RPE-1 and HuH7 cells were performed 48 h or 72 h prior to analysis using X-tremeGENE while those of Ts, HeLa, and CHO cells were done with Turbofect (ThermoFischer, Illkirch, France), according to the manufacturer’s instructions. Note that the HHL16 cell line was not used in these experiments because they are hard to transfect. Plasmids were used alone or up to a combination of six but the maximal amount of total DNA used per well (6-well plate, 50% confluence) never exceeded 1.5 µg. The amounts of plasmids were determined from the measurements of the efficacy of transfection of the combination involving the six plasmids of interest (SEPT2, SEPT6, SEPT7, SEPT9_i1 or SEPT9_i3, TTLL5, and TTLL11; Fig. S1). Importantly, the same individual amounts were used in every transfection experiment even when the plasmids were used alone. The pEGFP plasmid (Addgene) was used as a negative control. GFP-TTL, CFP-TTLL5, and CFP-TTLL11 expression vectors were kind gifts of Dr. C. Janke (Institut Curie, Orsay, France). GFP- and His-tagged septins (SEPT2, 6, 7 or 9_i3) and V5-SEPT9_i1 were kindly provided by Pr W. Trimble (Hospital for Sick Children, Toronto, Canada) and Dr. A. Gassama (CHB Paul Brousse, Villejuif, France), respectively.