Wild-type and KH3-4 domain mutant human IGF2BP2 sequences were PCR-amplified from corresponding pcDNA3-based vectors gifted by Hüttelmaier (Martin Luther University, Germany) with an HA-tag coding sequence inserted before the start codon, and subcloned into the pCDH lentiviral vector (CD513B-1, System Biosciences) through the XbaI and EcoRI enzymatic sites or into the pSIN4 vector. GPT2 cDNA ORF with an N-tenninal HA tag (HG22770-NY) was purchased from Sino Biological and subcloned into the pCDH lentiviral vector through the XbaI and NotI enzymatic sites. Human SLC1A5 coding sequence was reverse transcribed and PCR-amplified from 293T total RNA, with an N-tenninal HA tag being added during PCR, and cloned into the pCDH lentiviral vector through the XbaI and EcoRI enzymatic sites. shRNA vectors targeting human or mouse IGF2BP2, MYC, GPT2, and SLC1A5 were either purchased from GE Dharmacon or constructed by synthesizing shRNA-encoded DNA oligos and cloning into the pLKO.1 vector (Addgene). Mature antisense sequences of shRNAs were listed in Supplementary Table S1 .
Shrna vectors
Manufactured by Horizon Discovery
ShRNA vectors are molecular tools used for gene knockdown experiments. They contain short hairpin RNA (shRNA) sequences designed to target and silence specific genes within cells.
Automatically generated - may contain errors
2 protocols using shrna vectors
1
Lentiviral Cloning of Human Proteins
2
Cloning of Vps51, Vps13B, and Stx6/13 constructs
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Mouse full-length Vps51 cDNA was cloned into pGFP plasmid. For generating mouse full-length Vps13B cDNA constructs fused with GFP, cDNAs were amplified as four different fragments with over 20 bp overlap region each other from cDNA made from mouse liver total RNA, and the fragments and pEGFP plasmid (pEGFP-N1 or pEGFP-C1) were assembled using the HiFi DNA Assembly cloning kit (New England Biolabs) (all the primer sequences used in this study are provided in Supplementary Table 2 ). For the construction of the Vps51-GFP-MAO or Vps13B-GFP-MAO expression vector, human full-length Vps51 or Vps13B and human monoamine oxidase B (MAO-B) C-terminal TMD (469–520) were cloned into pEGFP-N vector (Clontech). shRNA vectors of Vps51 and Vps13B were from GE Dharmacon. Rat Stx6and rat Stx13 cDNA was subcloned into pEGFP-C vector. All clones were verified by sequencing.
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