Mammalian expression vector

The pcDNA3.1 (-) Mammalian Expression Vector (Invitrogen, Cat # V79520) was digested with the NheI (Thermo Fisher Scientific, Cat # FD0974) and the MssI (PmeI, Thermo Fisher Scientific, Cat # ER1341) restriction enzymes. After, fragments (IDT gene blocks) bearing the human codon-optimized (IDT tool) super folder green fluorescent protein^{54 (link)} (sfGFP) coding sequence and a synthetic 3’ UTR containing twelve version-six MS2 bacteriophage stem-loops (12XMBSV6^{16 (link)}) were cloned into the digested pcDNA3.1 (-) vector (Thermo Fisher Scientific, Cat # V79520) via Gibson assembly^{55 (link)}.

The coding sequence of the candidate PtGFT was used to prepare a DNA construct in the pcDNA^TM3.1⁽⁺⁾ Mammalian Expression Vector (Invitrogen, Life Technologies). The PtGFT coding sequence from the NCBI accession no. XM_002177440.1 (nucleotides 39–1121) in fusion with the nucleotide sequence encoding a HA-tag at its 5′ end and a “GCCACC” Kozak sequence was obtained by gene synthesis and then cloned as a HindIII-XhoI fragment in the pcDNA^TM3.1^(+).plasmid. The synthetic DNA sequence is registered under the NCBI accession number KT737477. Transient expression of PtGFT gene in CHO-gmt5 cell line, immunodetection of proteins and affinostaining with Aleuria Aurantia Lectin (AAL), were carried out as previously reported in Zhang et al. (2012) (link).

The full-length coding sequences of wild-type human FAK (GenBank accession no. L13616.1) and Src (GenBank accession no. NM_005417.4), including start and stop codons, were amplified from human hypertrophic scar-derived fibroblast cDNA by PCR using primers listed in Table 2, and they were individually cloned into the KpnI/XhoI sites of the pcDNA3.1(+) Mammalian Expression Vector (Invitrogen). The mutation of FAK-Tyr⁴⁰⁷ or Src-Tyr⁵²⁹ to phenylalanine (Phe) or glutamic acid (Glu), respectively, was performed by three-step mutagenic PCR29 , using the wild-type FAK or Src construct as the template. Each desired mutation was introduced by the amplification of two overlapping portions of FAK or Src using two primer pairs (a mutagenic primer paired with a flanking primer with the opposite orientation; Table 2). The two PCR products were then fused together by overlap extension29 , from which the full-length cDNA was amplified with primers listed in Table 2. The cDNA encoding the full-length FAK, Src, or their corresponding mutant FAK Y407F or Src Y529E was individually cloned into pcDNA3.1(+) Mammalian Expression Vector. All sequences were verified by direct DNA sequencing to confirm the full-length and mutant cDNA constructs. For the transfection into hypertrophic scar-derived fibroblasts, plasmid DNA was prepared with a HiSpeed Plasmid Midi Kit (Qiagen, Hilden, Germany).