Pcmv myc n vector

Mouse Prdx V expression (WT, C48S, C73S, C152S, and C48/152S) vectors with N-terminal HA-tags were constructed as in our previous study [24 (link)]. The full-length DBT cDNA was purchased from Korea Human Gene Bank (clone ID: hMU001786). The human DBT was subcloned into the pCMV-Myc-N vector with N-terminal myc-tag (Clontech Laboratories, CA, USA).

The IBDV VP2 gene from segment A was cloned into the pCMV-Flag-N vector (635688; Clontech, Mountain View, CA) and the pCMV-Myc-N vector (635689; Clontech, Mountain View, CA). All mutants of pCMV-Flag-VP2 and rescue plasmid T7-A were constructed by site-directed mutagenesis technology. Expression plasmid pEGFP-LC3B was constructed in our laboratory (33 (link)). The human p62 gene was amplified by PCR from total cellular RNA with gene-specific primers (5′-GCGAATTCGCATGGCGTCGCTCACC-3′ and 5′-GCTCTAGAGCTCACAACGGCGGGGG-3′) and subcloned into the pCMV-Flag-N vector and the pCMV-Myc-N vector. The mutants of Myc-p62, Myc-p62△UBA, and Myc-p62-△LIR were constructed with special primers. The primers 5′-TTGTACCCACATCTCCCCCCGCCGTTGTGA-3′ and 5-TCACAACGGCGGGGGGAGATGTGGGTACAA-3′ were used for Myc-p62△UBA. The primers 5′-GAGGAGATGATGACTCTTCAAAAGAAGT-3′ and 5′-ACTTCTTTTGAAGAGTCATCATCTCCTC-3′ were used for Myc-p62△LIR. HA-Ub, HA-K48, and HA-K63 expression plasmids were kindly gifted by Hongbin Shu (College of Life Sciences, Wuhan University). All expression plasmids were transfected into 293T cells and DF-1 cells using Lipofectamine 3000 reagent (L3000015; Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Total RNA was extracted from the patient’s blood using RNeasy Kits (QIAGEN, Hilden, Germany) and reverse-transcribed using SuperScript 2 reverse transcriptase (Thermo Scientific, Massachusetts, USA) and oligo (dT) primers. Amplification of the coding region of the FBP1 gene was carried out using the forward primer 5′-CACCATGGCTGACCAGGCGCCCTTCG-3′ and the reverse primer 5′-TCACTGGGCAGAGTGCTTCTCATAC-3′. The PCR procedure consisted of the following steps: (a) denaturing at 98 °C for 2 min, followed by 94 °C for 15 secs; and (b) annealing for 30 secs at 58 °C and extension at 72 °C for 1 min for 30 cycles. The PCR products were subcloned into the pGEM-T Easy Vector (Promega, Wisconsin, USA). Then, the FBP1 fragments (WT and 2 mutants) were cut from the pGEM-T Easy Vector using EcoRI and subcloned into a pCMV-Myc-N Vector (Clontech, California, USA). A corresponding pCMV-Myc-N Vector was used as a negative control.