SFB-ZRANB1 and its deletion-mutant-containing expression vectors were described in our previous publication (Zhang et al., 2018a (link)). pCLXSU (GFP)-HA-ZRANB1 and its C443A mutation were gifts from Shao-Cong Sun’s laboratory (Houston, TX, USA) stock as described previously (Jin et al., 2016 (link)). Full-length ZRANB1, which was obtained from the HA-ZRANB1 expression vector, was subcloned into the pCDH-MYC-S-tag and pCDH-FLAG vectors, and its C443A mutation was subcloned into the pCDH-MYC-S-tag vector. The SLC7A11-FLAG-HA-pHAGE vector was obtained from Alex Toker’s lab (Boston, MA, USA; Lien et al., 2017 (link)). Full-length SLC7A11 was subcloned into the pBabe-SFB and pLenti-V5 vectors using the Gateway system (Invitrogen) or into the pCDH-MYC-S-tag and pHAGE-MYC vectors using the HiFi system (NEB; New England Biolabs). pRK5-HA-ubiquitin was obtained from Addgene (plasmid number: 17608). The SLC7A11 shRNAs were subcloned into pLKO vectors and the sequences were as follows: shSLC-1:5′-CCGGGCTCTCATTTAAGGTTCCCTTCTCGAGAAGGGAACCTTAAATGAGAGCTTTTTG-3′; shSLC-2: 5′-CCGGCCTACATCATCGGTACTTCAACTCGAGTTGAAGTACCGATGATGTAGGTTTTTG-3′.
Hifi system
Manufactured by New England Biolabs
Sourced in United States
The HiFi system is a high-fidelity DNA amplification tool designed for accurate and reliable nucleic acid replication. It provides consistent and efficient DNA amplification for a variety of applications.
Automatically generated - may contain errors
3 protocols using hifi system
1
ZRANB1 and SLC7A11 Expression Vectors
2
Development of DDIT4 and APEX2 Fusion Proteins
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The murine DDIT4 CDS+3′UTR, human DDIT4 CDS, and APEX2 CDS (without FLAG and NES tags) were amplified by PCR using the proofreading Phusion polymerase (ThermoFisher) from Neuro-2a cells, SK-N-SH cells, and pcDNA3 FLAG-APEX2-NES plasmid (Addgene #49386), respectively (see Table S2 for primer sequences). The PCR products were then cloned between the HindIII and BamHI restriction sites on the paavCAG-pre-mGRASP plasmid (Addgene #34911) using the HiFi system (NEB, Massachusetts, CA, USA). Two alternative DDIT4 fusion proteins were prepared that contained the full-length sequences of DDIT4 and APEX2 and either a short flexible (GGGS)3 or rigid (GGAEAAAKEAAAKAAPAEAAAKEAAAKA) linker sequence in between. Sanger sequencing verified the DNA sequence of all constructs at CeMIA SA (Larisa, Greece).
3
Engineered TIA1-APEX2 Fusion Proteins
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We amplified the human TIA1 CDS and APEX2 CDS (without FLAG and NES sequences) by PCR, using the proofreading Phusion polymerase (ThermoFisher) (for primer sequences, see Supplementary Table S1 ) from human SK-N-SH cells and pcDNA3 FLAG-APEX2-NES plasmid (Addgene # 49386), respectively. The PCR products were cloned using the HiFi system (NEB, Ipswich, MA, USA) between the HindIII and BamHI restriction sites of the paavCAG-pre-mGRASP plasmid (Addgene # 34911). We prepared three TIA1 fusion proteins that contained the full-length sequences of TIA1 and APEX2 with either short flexible (flexible 1: (GGGS)3 and flexible 2: GSAGSAAGSGEF) or rigid (GGAEAAAKEAAAKAAPAEAAAKEAAAKA) linker sequences in between. Sanger-sequencing verified the DNA sequence of all constructs (CeMIA SA, Larisa, Greece).
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