P3 flag cmv 10 vector

The full-length GFP-HDAC6 was purchased from OriGene Technologies, and p3xFLAG-parkin WT (FLAG-parkin) plasmid was a gift from Dong Hyun Sohn (Pusan University, Korea). Four multiple domains of parkin were constructed by inserting the cDNA into the p3xFLAG-CMV10 vector. Briefly, the FLAG-parkin and its deletion mutants (1–3) were constructed by inserting cDNA into the p3×FLAG-CMV10 vector (Sigma) between EcoRI and EcoRV sites. The multiple domain site mutants (Mt1, Mt2, or Mt3) of parkin were generated using the full-length FLAG-parkin plasmid as the template (see Additional file 1: Table S1 for detailed information). For cell transfection of plasmids, the transfection mixture used to transiently transfect the described cell lines was based on a mixture of plasmid DNA and Lipofectamine 3000 transfection reagent, as described in the procedures (Thermo Fisher Scientific). Plasmid DNA and transfection reagent were dissolved in α-MEM (without additives) and incubated at room temperature for 30 min. The mixture was added dropwise into the cell culture dish and incubated for 24 h in HEK 293 cells or further cultured for 3 days (including M-CSF and RANKL) in RAW 264.7 cells at 37 °C under an atmosphere of 5% CO₂.

The PPRV N protein cDNA (GenBank accession no. HQ197753.1) was cloned into the mammalian expression vector p3 ×Flag-CMV10 vector (Sigma) with the EcoRI and KpnI restriction enzymes to generate the p3 ×Flag-N plasmid. The pHA-NCL plasmid encoding the NCL protein with a HA tag was obtained by cloning NCL cDNA (GenBank accession no.XM_012163051.2) into a pCMV-HA vector (Clontech). For prokaryotic expression of the glutathione S-transferase (GST)-tagged N protein, DNA encoding the N protein was subcloned into the pGEX-4T-1 vector (Invitrogen) with the EcoRI and NotI restriction endonucleases. Truncated mutants of NCL and PPRV N were generated from pHA-NCL and p3 ×Flag-N by conventional PCR (the primers will be made available upon request). All constructs were confirmed by sequencing (Sangon Biotech).

The swine eEF1A gene (GenBank accession no. NM_001097418.2) and its various domains were amplified by PCR and cloned into the pMyc vector (Clontech, Palo Alto, CA, USA) to generate pMyc-eEF1A, pMyc-eEF1A(1-333), pMyc-eEF1A(1-237), pMyc-eEF1A(238-462) and pCMV-Myc-eEF1A(201-333), respectively. The CSFV NS5A gene (GenBank accession no. EU497410.1) was amplified by PCR and cloned into the p3×Flag-CMV-10 vector (Sigma-Aldrich, ST. Louis, MO, USA) or pCMV-Myc to generate p3×Flag-NS5A or pMyc-NS5A. The primers for amplifying eEF1A and NS5A genes are listed in Table 1.

FLAG-UBR5 (#37188), HA-GSK3β (#14753), HA-GSK3β-S9A (#14754), and HA-GSK3β-K85A expression plasmids were from Addgene (USA). FLAG-AMPKα1 (CH805185), FLAG-SKP2 (CH896343), and FLAG-AKT1 (CH846646) were purchase from Vigenebio (Shangdong, China). SIRT7 and GSK3β deletion mutants were constructed into the p3× FLAG-CMV10 vector (Sigma). GST-tagged SIRT7 or SIRT7-S259A/Thr263A/Thr255A was constructed by cloning into pGEX-4T-3 (GE Healthcare). The other SIRT7 mutants were generated by the site-directed mutagenesis using the KOD-Plus-Neo DNA polymerase (TOYOBO). All plasmids were confirmed by DNA sequencing (Genewiz). Details of the primers used for the generation of these plasmids or other experiments are shown in Supplementary Table 2.