Feline Lindhout

130 4 transferred back to their original medium. Cells were maintained for 4 days (for knockdown experiments) or 24–48 h (for other experiments) prior to fixation or live-cell imaging. Cell line culture and transfection COS7 and HEK293T cells were cultured on plastic at 37°C in 5% CO2 in DMEM/Ham’s F10 (50%/50%) medium supplemented with 10% FCS and 1% penicillin/streptomycin. COS7 and HEK293T cells were plated on, respectively, 18-mm glass coverslips or plastic 1 day prior to transfection with MaxPEI (Polysciences). In brief, MaxPEI/DNA (3:1 ratio) was mixed in fresh serum-free DMEM or Ham’s F10 medium, incubated for 20 min, and added to the cell culture. After 24 h, cells were either processed for biochemistry, fixed, or used for live-cell imaging. DNA plasmids The following plasmids are described previously: Myc-VAPA, Myc-VAPB, HA-VAPA (with E178G mutation), HA-VAPB, HA-VAPB-K87D/M89D, GFP-VAPB-TM, GFP-VAPB-MSP- CC, and VAPB-MSP-GFP (with E6I single nucleotide polymorphism and K139R mutation; Teuling et al, 2007); pGFPC1-Sec61β (Hradsky et al, 2011); HA-BirA (de Boer et al, 2003); BioGFP (Jaworski et al, 2009); GW1-RFP and pGW1-GFP (Hoogenraad et al, 2005); pSuper vector (Brummelkamp et al, 2002); ssRFP-KDEL (Addgene plasmid #62236, gift from Dr. Erik Snapp; Snapp et al, 2006), TagRFP-ER (Schatzle et al, 2018), HA-Erlin1, and HA-Erlin2 (gift from Dr. Richard J.H. Wojcikiewicz; Pearce et al, 2007, 2009); GCaMP6f (Addgene plasmid #58514, gift from Prof. Adam E. Cohen; Venkatachalam & Cohen, 2014); and R-GECO1 (Addgene plasmid #45494, gift from Prof. Robert E. Campbell; Wu et al, 2013). The cDNAs of SCRN1a (AAH_40492.1), SCRN2 (AAH_10408.2), and SCRN3 (AAI_19685.1) were obtained from a human cDNA library kindly provided by Dr. Mike Boxem. All wild-type SCRN1, SCRN1-N (1–293), SCRN1-C (293–414), SCRN2, and SCRN3 constructs were generated using PCR-based cloning strategies and inserted into β-actin (for HA-SCRN1) or GW1 (for all other constructs) vectors. Constructs with single- point mutations were generated using site-directed mutagenesis. SCRN1 FFAT-like mutant constructs were obtained for each predicted FFAT-like motif identified by a previously reported algorithm (Murphy & Levine, 2016). More specifically, SCRN1-Y40A, SCRN1- F144A, SCRN1-F153A, and SCRN1-F402A were generated by replacing the conserved hydrophobic phenylalanine or tyrosine residue for an alanine residue. Proteolytic dead mutant constructs SCRN1-C9A, SCRN2-C12A, and SCRN3-C6A were generated by replacing the predicted proteolytic cysteine residue, as identified by the online MEROPS database, by a non-catalytic alanine residue. The RIM1a-mCherry construct was obtained by exchanging the HA-tag of the previously reported pAJ14063-pFUGW-RIM1aWT-HA construct (de Jong et al, 2018). The following shRNAs inserted in pSuper vectors were used in this study: VAPA shRNA #1 (5′-GCATGCAGAGTGCTGTTTC-3′; Teuling et al, 2007),