The IncE sequence used in this study is from the L3 serovar L3/404/LN (NCBI reference WP_015506602) (Harris et al., 2012 (link)). The pGEX-4T-2 bacterial expression plasmid encoding the human SNX5 PX domain (residues 22–170) was generated using a standard PCR-based cloning strategy, and its identity confirmed by sequencing. All other bacterial expression constructs for human SNX proteins were synthesized and cloned into pGEX-4T-2 by Genscript (USA). These included the SNX5 PX domain IncE fusion (SNX5 residues 22–170 with IncE residues 108–132 fused at the C-terminus (Figure 4—figure supplement 1A ), SNX6 (residues 29–170), SNX32 (residues 17–166), and SNX5 PX domain mutants. The pcDNA3.1-N-eGFP mammalian expression constructs encoding full-length human SNX5, SNX5(F136A), IncE(91-132) and IncE(91-132)(F116D) with N-terminal GFP-tags were generated by Genscript (USA). The pCMU-myc-SNX5 was as described previously (Kerr et al., 2006 (link)), and the SNX6 and SNX32 genes cloned into the pcDNA3.1-nMyc vector at BamHI and XhoI restriction sites (Kerr et al., 2012 ). SNX5, SNX32 and SNX8 were also cloned by polymerase chain reaction, restriction digest and ligation into pEGFP-C1 for expression with N-terminal GFP tags as described previously (Wang et al., 2010 (link)).
Pcdna3.1 n egfp
Manufactured by GenScript
Sourced in United States
PcDNA3.1(+)-N-eGFP is a plasmid vector that enables the expression of the enhanced green fluorescent protein (eGFP) gene under the control of the CMV promoter. This plasmid can be used for transient transfection and gene expression studies in various cell lines.
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Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pcdna3.1 vector, by GenScript (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Egfp c 1, by GenScript (2 mentions)
Easy peptm mini ms sample prep kit, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Merck Group (1 mentions)
Spntm protein assay kit, by G Biosciences (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Pmcherry c1, by Takara Bio (1 mentions)
Quinalizarin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Cos 7, by American Type Culture Collection (1 mentions)
Primestar mutagenesis basal kit, by Takara Bio (1 mentions)
Nunc cell culture dishes, by Thermo Fisher Scientific (1 mentions)
Transit lt1 transfection reagent, by Mirus Bio (1 mentions)
H33342, by Thermo Fisher Scientific (1 mentions)
10 protocols using pcdna3.1 n egfp
1
Cloning and Expression of Sorting Nexin Proteins
2
Mfn2 Silencing and Rescue in Cells
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Mfn2 silencing was achieved by using a validated pool of siRNA duplexes directed against mouse Mfn2 (TriFECTa Kit, IDT) and Lipofectamine RNAiMAX Transfection Reagents (Invitrogen) according to the manufacturer's instructions (see Figure 3—source data 1 for the dicer-substrate short interfering RNA, DsiRNA, sequence). Cells given scrambled siRNA were used as a negative control. To rescue siRNA knockdowns, a siRNA-resistant cDNA that expresses wildtype Mfn2 was cloned in the pcDNA3.1+vector (GenScript) under a constitutive CMV promoter. The codon was optimized to be resistant to the siRNA added (see Figure 3—source data 1 for cDNA sequence). The control vector was pcDNA3.1+N eGFP (GenScript), which expresses GFP instead of Mfn2. For rescue experiments, cells were first knocked down with siRNA for 12 hr and then transfected with plasmids using Lipofectamine 3000 (Invitrogen) for 36–48 hr. For overexpression, wildtype cells were transfected with plasmids for 36–48 hr.
3
Proteomic Analysis of A549 Cells
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The proteomic analysis described in this study was performed on A549 cells transfected with the empty vector pCDNA3.1(+)N-EGFP (Genscript, Piscataway, NJ, USA) or with the same cells albeit those expressing the Np protein (pCDNA3.1(+)N-EGFP-NP). Expression at 72 h after transfection was verified by Western blot (Supplementary Figure S1A ). For each condition, about 2 × 106 cells were used and three biological replicates were examined. After determining the protein concentration with the SPNTM-Protein assay kit (G-Biosciences, St. Louis, MO, USA), 50 µg of the resulting suspensions were lysed, reduced/alkylated and enzymatically digested with Trypsin and Lys-C using the Easy PepTM Mini MS Sample Prep Kit (Thermo Scientific, Rockford, IL, USA). Following the kit protocol, in less than 3 h and for each examined condition, peptides were generated, cleaned-up to prepare detergent-free samples and resuspended in 0.1% formic acid (Sigma, St. Louis, MO, USA) for LC-MS/MS analysis.
4
Construction of Fluorescently-Tagged Proteins
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Coding sequences of IMP1, IMP2, IMP3, ELAVL2, STAU1 and FMRP were PCR-amplified and cloned into pEGFP-C1 (Clontech), and PABPC1 coding sequence was cloned into pEGFP-N1 (Clontech) by restriction enzyme digestion. GFP-IMP1KH1-4mut construct was obtained by site-directed mutagenesis of the GXXG loops in the four KH domains (17 (link)) from GK(E/K/G)G to GELG. YBX1 was cloned into pcDNA3.1 + N-eGFP (Genscript) inserting a 25 aminoacid flexible linker (5× GGGGS) (18 (link)) between the fluorescent tag and YBX1. IMP1 and YBX1 (including flexible linker) coding sequences were also cloned into pmCherry-C1 (Clontech). EGFP was PCR-amplified from pEGFP-C1 and cloned into mCherry-C1 by restriction enzyme digestion in order to obtain mCherry-GFP fusion protein. 2xGFP oligomer was obtained by cloning an EGFP sequence with a flexible linker (3xGGGGS) upfront into a pEGFP-C1 vector. 5xGFP oligomer was obtained by cloning a 4xGFP sequence with 3xGGGGS flexible linkers in between each EGFP unit into a pEGFP-C1 vector by restriction enzyme digestion.
5
Silencing and Overexpression of CPT1A
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CPT1A silencing was achieved by using a validated pool of small interfering RNA (siRNA) duplexes directed against human CPT1A (Trifekta Kit, IDT) and Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer’s instructions (see S1 Text for the dicer-substrate short interfering RNA [DsiRNA] sequence) [19 (link)]. The knockdown (KD) efficiency was determined by measuring CPT1A mRNA levels with a premade primer (IDT) and quantitative RT-PCR (Applied Biosystems). The expression levels were normalized to an HPRT endogenous control. Cells given scrambled siRNA were used as a negative control. For overexpression of human CPT1A, the cDNA was cloned in the pcDNA3.1+ vector (GenScript) under a constitutive CMV promoter. The codon was optimized to be resistant to the siRNA added. The catalytically dead CPT1A had an identical sequence (see S1 Text ) to the wild-type siRNA-resistant CPT1A, with the exception of G709E and G710E mutations to abolish catalytic activity (GenScript). For transduction, CPT1A was first knocked down with Lipofectamine RNAiMAX for 24 hours. Next, cells were transduced with plasmids using Lipofectamine 3000 (Invitrogen) for 4 hours. Media were then refreshed, and cells were assayed 48 hours post plasmid transduction (72 hours post siRNA knockdown). The control vector was pcDNA3.1+N-eGFP (GenScript), which expresses GFP instead of CPT1A.
6
Antibody and Plasmid Generation
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Anti-actin (3700) and anti-α/β tubulin (2148) antibodies were purchased from Cell Signaling Technology. Anti-ENO1 (ab155102) and pan-phospho-serine (ab9332) antibodies were purchased from Abcam. Anti-V5 (R960-25) antibody and streptavidin-FITC conjugate (S-869) were purchased from Thermo Fisher Scientific. Anti-GFP (11, 814, 460, 001) antibody was purchased from Sigma-Aldrich. Li-Cor IRDYe fluorescent secondary antibodies 680-anti-rabbit (926-68071) and 800-anti-mouse (926-32210) were purchased from Millennium Science. D4476 casein kinase I inhibitor was purchased from Abcam. Leptomycin B was purchased from BioAustralis. Quinalizarin, protease inhibitor cocktail, phosStop inhibitor and other chemicals were purchased from Sigma-Aldrich, or as otherwise specified. The plasmids encoding GFP-ENO1, GFP-ENO1-S419A and GFP-ENO1-S419D were generated by GenScript in pcDNA 3.1-N-eGFP. Plasmids encoding V5-miniTurbo-NES were obtained from Professor Mike Ryan (Monash University), mutagenesis resulting in removal of the NES sequence and insertion of ENO1's sequence was performed by GenScript.
7
Glycine Transporter Expression in Cos-7 Cells
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Cercopithecus aethiops SV40-transformed kidney (Cos-7) cells were obtained from American Type Culture Collection (ATCC CRL-1651) and cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. The cells were passaged every 2–4 d. For electrophysiological experiments, the cells were seeded into 35-mm dishes. Once confluent, they were transfected with a plasmid encoding N-terminally GFP-tagged hGlyT1b or hGlyT2a, respectively; the cDNAs of hGlyT1b in eGFP-C-1 and hGlyT2b in pcDNA3.1(+)-N-eGFP were bought from Genscript. The transfection was performed with jetPRIME (0.8 µg DNA/dish) according to the manufacturer’s protocol. On the following day, the cells were seeded at low density into poly-D-lysine–coated dishes. Current recordings were conducted 16–24 h after seeding.
8
CRISPR/CasRx System for Gene Editing
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Sense and antisense Dock5 and Elmo2 guide(g)RNAs (Fasmac, Atsugi, Japan) were annealed and inserted into the mammalian small RNA transcription vector pSINsi-mU6 (Takara Bio, Kyoto, Japan, Takara Bio Gene No. X06980) in accordance with the manufacturer’s instructions. The mammalian expression plasmid encoding CasRx, integrated into the clustered regularly interspaced short palindromic repeats (CRISPR)/CasRx system, was purchased from Addgene (Watertown, MA, USA; Addgene Gene No. 109049). The isolated N-terminal region (amino acids 1 to 71) of human Dock5 was chemically synthesized and fused with the mammalian expression vector pcDNA3.1-N-eGFP (GenScript, Piscataway, NJ, USA).
The R676C mutations of Sema5A were generated from the plasmids encoding mouse Sema5A (Addgene Gene No. 72161) using the PrimeStar Mutagenesis Basal kit (Takara Bio) in accordance with the manufacturer’s instructions [36 (link)].
The R676C mutations of Sema5A were generated from the plasmids encoding mouse Sema5A (Addgene Gene No. 72161) using the PrimeStar Mutagenesis Basal kit (Takara Bio) in accordance with the manufacturer’s instructions [36 (link)].
9
Stable Expression of hSERT-GFP in HEK293
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hSERT tagged with GFP was stably expressed in a tetracycline-inducible HEK293 cell line. HEK293 cells have been previously authenticated by short tandem repeat profiling at the Medical University of Graz (Cell Culture Core Facility, Graz, Austria). Cercopithecus aethiops SV40-transformed kidney (Cos-7) cells were obtained from American Type Culture Collection (CRL-1651; Manassas, VA). Cos-7 were transfected with a plasmid encoding N-terminally GFP-tagged hGlyT1b or hGlyT2a, respectively; the complementary DNAs of hGlyT1b in eGFP-C-1 and hGlyT2b in pcDNA3.1(+)-N-eGFP were bought from GenScript (New York, NY). The transfection was performed with jetPRIME (0.8 μg DNA/dish) according to the manufacturer’s protocol. All cells were maintained in Dulbecco's Modified Eagle’s Medium containing 10% fetal bovine serum. Twenty-four hours before the experiment, the cells were seeded at low density onto poly-D-lysine-coated dishes (35 mm Nunc Cell culture dishes, Thermo Fisher Scientific, Waltham, MA).
10
Transfection and Imaging of GFP-Actin Fusion
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Protein constructs were synthesized with an N-terminus GFP in pcDNA3.1(+)-N-eGFP (GenScript). Actin staining was performed with rhodamine-phalloidin (TRITC) (ThermoFisher R415) at 1:200/500 dilution and nuclei stained with H33342 (ThermoFisher 62249) at 1 µg/mL U2OS (human osteosarcoma) cells were cultured in high glucose Dulbecco’s modified Eagle’s (DME) media with 4,500 mg/L glucose, supplemented with 10% fetal bovine serum (FBS) (HyClone). Cells were grown at 37 °C in an incubator filled with 5% CO2 and 99% humidity. U2OS cells seeded onto 22 × 22-mm glass coverslips in 35-mm culture dishes and grown to subconfluence. Each dish was then transfected with 0.5 μg of GFP control of fusion gelsolin construct plasmid DNA using the Mirus TransIT LT1 transfection reagent according to the manufacturer’s protocol, incubated at 37 °C and allowed to express for 24 h. Cells were then fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min.
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