Full-length human CD63 was subcloned from CD63-pEGFP C2 (a gift from Paul Luzio, Addgene plasmid #62964) to pcDNA™6/myc-His A vector (Invitrogen, Waltham, MA, USA) to produce CD63 without tag using KpnI–BamHI enzymes. Then, mCherry sequence was obtained by PCR from the plasmid mCherry-hALIX (a gift from James Hurley, Addgene plasmid # 21504 [46 (link)] using 5′-GGG TAC CAT GGT GAG CAA GGG CGA GGA G (forward) and 5′-GGC ATG GAC GAG CTG TAC AAG TGG TAC CCC (reverse) primers and cloned into the pcDNA™6/myc-His A vector (Invitrogen) in frame with CD63 to produce mCherry-CD63 using KpnI enzyme. The previously mentioned mCherry sequence was also cloned into pcDNA™6/myc-His A vector to produce mCherry protein alone.
Pcdna6 myc his a vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PcDNA6/myc-His A vector is a commercially available plasmid vector designed for protein expression and detection in mammalian cells. It provides a strong cytomegalovirus (CMV) promoter for high-level transgene expression and a C-terminal myc and 6xHis tag for protein purification and detection.
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Lab products found in correlation
Oligofectamine transfection reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
P3xflag cmv vector, by Merck Group (1 mentions)
P3xflag cmv 10, by Merck Group (1 mentions)
Kod dna polymerase, by Toyobo (1 mentions)
Pcdh cmv mcs ef1 puro vector, by System Biosciences (1 mentions)
Pgex 6p 1 vector, by GE Healthcare (1 mentions)
Pflag cmv5 vector, by Merck Group (1 mentions)
Pgex 4t 1, by GE Healthcare (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Endofree plasmid maxi kit, by Qiagen (1 mentions)
Mcherry halix, by Addgene (1 mentions)
Cd63 pegfp c2, by Addgene (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Nutrient mixture f 12, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
10 protocols using pcdna6 myc his a vector
1
Cloning mCherry-CD63 Fusion Protein
2
LINC00941 Silencing and Overexpression Effects on Cancer Cell Proliferation
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Cells were seeded in 96-well plates at 3 × 103 per well for MIA PaCa-2 and AsPC-1 or at 1 × 103 per well for PCI-35. Twenty-four hours after seeding, the cells were transfected with small interfering RNAs (siRNAs) for LINC00941 (si#1, sense: 5′-GCCUCCAUAUUCAUGAACUtt-3′, antisense: 5′-AGUUCAUGAAUAUGGAGGCtg-3′, si#2, sense: 5′-CCAUUCAGCCUUGAACAUUtt-3′, antisense: 5′-AAUGUUCAAGGCUGAAUGGtc-3′, Thermo Fisher Scientific, Waltham, MA, USA) or Silencer Select Negative Control (Thermo Fisher Scientific) at 200 nmol/L using Oligofectamine Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. Then, 24 h after transfection, the expression of LINC00941 was assayed by qRT-PCR. A colorimetric cell proliferation assay employing 0.05% MTT (Sigma) was carried out as described previously [12 (link)]. For the proliferation assay with forced expression of LINC00941, full-length LINC00941 cDNA was cloned into the pcDNA6/myc-HisA vector (Invitrogen). The cloned vectors were verified by DNA sequencing. The cells were transfected with the cloned vector or the empty vector at 1 ng/μL using Lipofectamine3000 Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. Finally, 24 h after transfection, the expression of LINC00941 was assayed by qRT-PCR, and an MTT assay was carried out in the same way.
3
Overexpression of Constitutively Active Fyn in HCASMCs
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pcDNA6-myc/His A FynYF was transfected as previously described.19 In brief, human cDNAs encoding the constitutively active form of Fyn with the Y530F mutation (ca-Fyn) were subcloned into the pcDNA6-myc/His A vector (Invitrogen, Thermo Fisher Scientific). The construct was verified by DNA sequencing.
HCASMCs were trypsinized, counted, and divided into at least 5 × 105 cells per tube when the cell confluence reached 90%–100%. An Amaxa Human AoSMC Nucleofector Kit (Lonza, Tokyo, Japan) was used for the nucleofection of pcDNA6-myc/His A FynYF. HCASMCs were transfected with 2 μg of pcDNA6-myc/His A or pcDNA6-myc/His A FynYF using a Nucleofector II device following the manufacturer's instructions for the kit. PmaxGFP from the kit was used to monitor the transfection efficiency (>80%). HCASMCs were serum starved for 24 hours after transfection for 48 hours. Next, the cells were treated with 30 μM hesperetin for 30 minutes. Immunoprecipitation and western blot were applied to analyze the activation of Fyn and ROK in HCASMCs.
HCASMCs were trypsinized, counted, and divided into at least 5 × 105 cells per tube when the cell confluence reached 90%–100%. An Amaxa Human AoSMC Nucleofector Kit (Lonza, Tokyo, Japan) was used for the nucleofection of pcDNA6-myc/His A FynYF. HCASMCs were transfected with 2 μg of pcDNA6-myc/His A or pcDNA6-myc/His A FynYF using a Nucleofector II device following the manufacturer's instructions for the kit. PmaxGFP from the kit was used to monitor the transfection efficiency (>80%). HCASMCs were serum starved for 24 hours after transfection for 48 hours. Next, the cells were treated with 30 μM hesperetin for 30 minutes. Immunoprecipitation and western blot were applied to analyze the activation of Fyn and ROK in HCASMCs.
4
Extrachromosomal V(D)J Recombination Assay
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Extrachromosomal V(D)J recombination assays were performed in Br3neo human fibroblastoid cells as described previously [24 (link)], using either pGG49 (signal-joint formation) or pGG51 (coding-joint formation) as the reporter [25 (link)]. Briefly, we transfected human fibroblast cells with murine full-length Rag1, murine full-length Rag2, and a plasmid reporter substrate and harvested the cells at 48-hours post-transfection. The plasmid DNA encodes the gene for ampicillin (Amp) resistance, and further encodes a chloramphenicol (Cam) resistance gene preceded by a transcriptional terminator flanked by recombination signal sequences. Proper recombination confers Cam resistance. The plasmid DNA was isolated from the harvested cells and transformed into bacteria. V(D)J recombination frequency was measured by comparing the number of AmpR/CamR colonies to the number of AmpR colonies [26 (link)]. Full-length Rag2 was transiently expressed using the p3xFLAG-CMV vector (Sigma). Wildtype Rag1, Rag1V779M, and Rag1R142* were transiently expressed using the pcDNA6-myc-hisA vector (Invitrogen). Expression of all proteins was confirmed by Western analysis.
5
Construction of Plasmids for TET1 Study
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Four DNA fragments were used for construction of the five plasmids indicated in Figure S1a; 3xFLAG from p3xFLAG‐CMV‐10 (Sigma, St. Louis, MO, USA), MBD (codons 144 through 230) from pcDNA‐MBD2 13 , TET1‐CDwt (codons 1418 through 2136) from pcDNA3b‐hTET1‐CD‐HA 10 , and TET1‐ CDmut (codons 1418 through 2136, D1674A) from pcDNA3b‐hTET1‐CDmut‐HA 10 . The latter two were kindly provided by Drs. J.U. Guo and H. Song (Johns Hopkins University School of Medicine). E. coli strain DH5αF' was used for plasmid preparation. Relevant fragments were PCR‐amplified using KOD DNA polymerase (Toyobo, Osaka, Japan) and were cloned into the pcDNA6/Myc‐His A vector (Invitrogen, Carlsbad, CA, USA). Nucleotide sequences of the plasmids were confirmed. Nucleotide sequences of the PCR primers are available upon request to the authors.
6
Generating Tagged ANG Constructs
7
Plasmid Construction for NF-kB Signaling
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The plasmids pReceiver-M14/Flag-human HOIP, pReceiver-M09/Myc-human HOIL-1L, pReceiver-M07/HA-human SHARPIN, pReceiver-M06/HA-human OTULIN, pGEX-4T-1/GST-HOIP (633-1072), pGEX-4T-1/GST-NEMO (257-346), and pGEX-6P-1/GST-TNFα (77-233) were described previously (Lee et al., 2019 (link)). pReceiver-M06/HA-human TSG101 and pReceiver-M06/HA-human SPATA2 were obtained from GeneCopoeia (USA). To generate variants of the plasmid pFlag-CMV5/HOIP (amino acid residues 1-480, 481-632, or 637-1072), each cDNA was amplified by polymerase chain reaction (PCR) using pReceiver-M14/Flag-human HOIP as a template and inserted into a pFlag-CMV5 vector (Sigma-Aldrich, USA). To construct variants of the plasmid pcDNA6/myc-His A-HOIL-1L (amino acid residues 1-129, 130-270, or 271-500), each cDNA was amplified by PCR with pReceiver-M09/Myc-human HOIL-1L as a template and inserted into pcDNA6/myc-His A vector (Invitrogen, USA). The variants of pcDNA3/HA-TSG101 (amino acid residues 1-216 or 271-390) were generated by PCR from pReceiver-M06/HA-human TSG101 and insertion into a pcDNA3/HA vector. An expression vector encoding His6-TSG101 was constructed by subcloning the cDNA of human TSG101 into pET28a/His6 vector. The vector construct encoding pCMV-3Tag-4A/Myc-TSG101 was constructed by inserting the cDNA of human TSG101 into a pCMV-3Tag-4A vector (Stratagene, USA).
8
Recombinant Protein Expression in Mammalian and Bacterial Systems
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To express proteins in RAW264.7, genes of interest were amplified and cloned into the pcDNA6/myc-His A vector (Invitrogen Corp., Carlsbad, CA, United States). To express recombinant proteins in Escherichia coli BL21 cells (Stratagene, San Diego, CA, United States), genes of interest were amplified and cloned into the pQE-2 (Qiaen, Venlo, Netherlands) or pGEX-4 T-1 (GE Healthcare, Little Chalfont, United Kingdom) vectors. BL21 strains were grown at 37°C in Luria-Bertani (LB) broth (Nacalai). PCR primers used to amplify genes of interest are listed in Table 1 and were purchased from Greiner Japan or Europhins Genomics. Constructs for transfection were purified using the EndoFree Plasmid Maxi Kit (Qiagen), according to the manufacturer’s instructions. Plasmid construct sequences were confirmed by DNA sequencing. Recombinant proteins were prepared with Ni-NTA agarose (Qiagen) or glutathione sepharose 4B (GE Healthcare), according to the manufacturer’s instructions. To prepare recombinant proteins for cell stimulation, buffers were exchanged with phosphate-buffered saline (PBS) through PD-10 desalting columns (GE Healthcare), and endotoxins were removed using endotoxin removal resin (Pierce, Rockford, IL, United States) according to the manufacturer’s instructions.
9
Recombinant Human HtrA1 Variants
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A full-length open-reading frame of the HTRA1 gene (NM_002775.4; 1440 base pairs) was cloned into an empty pcDNA6/myc-His A vector (Invitrogen, Carlsbad, CA, USA) between the BamHI and NotI sites (pHis/myc-HTRA1) using specific primers (forward: 5′-TAATGGATCCCCATGCAGA TCCCGCGC-3′; reversed: 5′-TAATGCGGCCGCGGGTCAA TTTCTTCGGG-3′). Expression construct for the recombinant human HtrA1 variant (pHis/myc-HTRA1-12insSer) was generated by site-directed mutagenesis (QuikChange Lightning Multi Site-Directed Mutagenesis Kit; Strategene, La Jolla, CA, USA) using specific primers (sense: 5′-GCGCC GCTCTTCTCCCGCTGTCCTTGCTGCTGCTGCTGGCGG-CG-3′; anti-sense: 5′-CGCCGCCAGCAGCAGCAGCAAGG ACAGCGGGAGAAGAGCGGCGC-3′) based on the expression construct for the recombinant human wild-type HtrA1 (pHis/myc-HTRA1). Sequences of both constructs were verified by direct sequencing.
10
Overexpressing HtrA1 in ARPE-19 Cells
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The human ARPE-19 cell line (CRL-2302, American Type Culture Collection) and primary human fetal RPE cells were maintained in Dulbecco's modified Eagle's medium and F-12 nutrient mixture (Gibco BRL, Rockville, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL) and 1 × penicillin/streptomycin (Gibco BRL) at 37 °C in 5% CO 2 . 22 ARPE-19 cells at 90% confluence were transfected with 4 μg HtrA1 expression constructs in 10 μl Lipofectamine-2000 (Invitrogen) and incubated for 12-48 h before further analysis. ARPE-19 cells transfected with empty pcDNA6/myc-His A vector (Invitrogen) were used as control.
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