Pdsred monomer n1

A plasmid encoding ubiquinol-cytochrome c reductase, 6.4 kDa subunit (UQCR11) was fused with AcGFP as previously described^{83 (link)}. A plasmid encoding NADH-ubiquinone oxidoreductase subunit B8 (NDUFB8) fused with AcGFP was constructed by subcloning human NDUFB8 into the mammalian expression vector pAcGFP1-N1 (Clontech, Palo Alto, CA, USA). The coding region of NDUFB8 was amplified from the cDNA of the human breast cancer cell line MCF7 using the following primer set:
NDUFB8, forward: 5′-CCCGAGCTCGCCATGGGCGCGGTGGCCAGGGCC-3′
NDUFB8, reverse: 5′-GGGGTACCCCGATCTCATAGTGAACCACCCGC-3′.
Plasmids encoding cytochrome c oxidase subunit 8 A (COX8A) or ATP synthase F1 subunit gamma (ATP5F1c) fused with DsRed-Monomer were constructed by changing the fluorophores from the previously constructed plasmids encoding proteins fused with AcGFP^{83 (link)}. The coding regions of the proteins were subcloned into the mammalian expression vector pDsRed-Monomer-N1 (Clontech). The transfection of expression vectors was performed 24 h after seeding the cells using FuGENE HD (Promega, Madison, WI, USA), according to the manufacturer’s instructions.

NF-κB promoter luciferase reporter plasmids were kindly provided by Drs. Hongbin Shu and Youhai Chen. pDsRed-monomer-N1 and pEGFP-N1 vectors were purchased from Clontech (USA). pRK5-flag vector was kindly provided by Dr. Shimin Hu. Human sorcin was originally cloned from HEK293T cells by RT-PCR using the specific primers (Sense: 5′-ATGGCGTACCCGGGGCATC-3′; Anti-sense: 5′-TTAAACACTCATGACACATTGAATGAAATC-3′) that were designed according to the gene sequence of sorcin in GenBank (Access ID: NM_003130.3). The pRK5-flag-sorcin, pEGFP-sorcin, and pDsRed-sorcin expression plasmids were constructed by standard molecular biology techniques. Human STAT3 was originally cloned from HEK293T cells by RT-PCR using the specific primers (Sense: 5′-ATGGCCCAATGGAATC-3′; Anti-sense: 5′-TCACATGGGGGAGGTAGCGC-3′) that were designed according to the gene sequence of STAT3 in GenBank (Access ID: NM_139276.2). The pRK5-flag-STAT3 and pEGFP-STAT3 expression plasmids were constructed by standard molecular biology techniques. All the primers were synthesized by Sangon Company (Shanghai, China).

Full-length mouse Podoplanin was cloned into pAcGFPN1 and pDsRedmonomerN1 (Clontech).

DNA sequence encoding the scFv recognizing transferrin receptor (TfR) was generated by PCR amplification, with scFv-expressing vector clones [26 (link)] as the template, using the following primers: 5′-GCG GCC CAG CCG GCC ATG G-3′ and 5′-CTT GCG GCC GCA CCT AGG ACG GTC AGC TT-3′. The SfiI/NotI-digested PCR products were subcloned into pTNH6-Haals [27 (link)] at the corresponding restriction sites, resulting in pTNH6-HaalsαTfR-#1-8, respectively (Additional file 1 Table S1). A BsiWI-fragment of pCAG-T7F [19 (link)] was inserted into the BsiWI site of pVITRO1-neo-mcs (InvivoGen), resulting in pVF#9. A BsrGI-fragment of pTNH6-HaalsαTfR-#5 was inserted into the BsrGI site of pVF#9, resulting in pVF#9-TfR.
Plasmids were linearized by restriction digestion with PvuI (NEB) before transfection. HAC donor CHO cells (8 × 10⁴/well in 24-well plates (Nunc)) were co-transfected with 0.3 μg each of pTNH6-H and pCAG-T7-F, and 0.25 μg of pDsRed-Monomer-N1 (Clontech) using Lipofectamine 2000 (Invitrogen). At 24 h after transfection, the cells were re-plated at low density and selected for 14 days with 800 μg/ml of G418 (Nacalai). Drug-resistant cells were recovered as a mixed population. HT1080 cells (2 × 10⁶/6 cm dish) were co-transfected with 0.4 μg each of pTNH6-H and pCAG-T7-F. After culture for 6 h, syncytium formation was tested under the microscope.

The cDNA of pB4GALT5 was amplified and sub-cloned into the vector pFlag-CMV-2 (Sigma-Aldrich) and pDsRed-Monomer-N1 (Clontech) to generate the fusion plasmid of B4GALT5-DsRed and Flag- B4GALT5. The GP5 gene of PRRSV JXwn06 was amplified and cloned into the vector pcDNA3.1 (Invitrogen) and pEGFP-C1 (Clontech) to generate the plasmid of GP5-Myc and GFP-GP5. Above constructs were verified by sequencing and the sequences of primers were supplied in Table 1.

The full-length Danio rerio panx1a wild type (WT) open reading frame (amino acids 1-416) was cloned into the enhanced yellow fluorescent protein plasmid (pEFYP-N1) expression vector (Clontech Laboratories Inc., Mountain View, CA, USA) as described [4 (link)]. For Förster Resonance Energy Transfer (FRET) analysis, the same sequence was cloned into pDsRed-monomer-N1 (Clontech Laboratories Inc., Mountain View, CA, USA). For protein interaction studies, panx1a WT and mutants were cloned into a pdTomato-His expression vector. For localization studies, ER and Golgi organelle markers tagged with DsRed2 were generated as described [21 (link)]. Mutagenesis was performed using the Q5 Hot Start Site-Directed Mutagenesis kit (New England Biolabs Inc., Boston, MA, USA) according to the manufacturer’s protocol. Oligonucleotides (Table 1) were designed using NEBaseChanger tool and synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). All mutations were confirmed by double-stranded DNA sequencing (Eurofins, MWG Operon LLC, Huntsville, AL, USA).