For the production of recombinant adenovirus, His/HP1α cDNA was subcloned from pcDNA 3.1/HisC (Invitrogen) and inserted into the pacAd5-CMV-K-N pA backbone. The HP1α-S92A and HP1α-S92D mutants were generated via the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) utilizing synthesized primers (Supplementary Table 1) (Integrated DNA Technologies) and verified via sequencing (Mayo Clinic Sequencing Core). Empty vector (EV), wild type (WT), and mutant plasmids were packaged into adenoviral particles and viral titer determined by the Viral Vector Core at the University of Iowa. Chromatin bridges and micronuclei were quantified in greater than 500 cells over three independent imaging experiments and statistical significance determined by a Student’s t-test in GraphPad Prism 7.
Pcdna3.1 his c
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PcDNA3.1/His C is a plasmid vector designed for the expression of recombinant proteins in mammalian cell lines. It includes a strong cytomegalovirus (CMV) promoter, a polyadenylation signal, and a polyhistidine (His) tag sequence for purification of the expressed protein.
Automatically generated - may contain errors
Lab products found in correlation
Pgfp 5 rs vector, by OriGene (2 mentions)
Puno1 mcd274, by InvivoGen (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Arrest in transfection reagent, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Sartorius (1 mentions)
Dulbecco modified eagle medium (dmem), by Cytiva (1 mentions)
Dulbecco s modified eagle s medium, by Cytiva (1 mentions)
Anti xpress antibody, by Thermo Fisher Scientific (1 mentions)
C2c12 cell, by American Type Culture Collection (1 mentions)
8 protocols using pcdna3.1 his c
1
Recombinant Adenovirus Production
2
Construction of Plasmids Expressing YBX1, YBX3, and SRSF Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To obtain pFL-YBX1 and pXpress-YBX1, the full-length YBX1 cDNA was PCR-amplified and inserted into the HindIII and BamHI sites of the p3XFLAG-CMV (Sigma-Aldrich) or into the BamHI and XhoI sites of the pcDNA3.1/HisC (Invitrogen), respectively. To generate pHA-YBX1 and pHA-YBX3, YBX1 and YBX3 cDNAs were amplified using 5′-terminal primers encoding two copies of the influenza hemagglutinin (HA) tag peptide and the amplified DNAs were inserted into the HindIII and BamHI sites of pcDNA3 (Invitrogen). The same approach was used for construction of pFL-YBX3 except that the 5′-terminal PCR primer encoded two copies of the Flag tag. To obtain pXpress-YBX1CSDm, appropriate point mutations were introduced into the pXpress-YBX1 expression plasmid by using PCR-mediated mutagenesis. To generate pFL-SRSF1, pFL-SRSF2, and pFL-SRSF3, full-length cDNAs of human SRSF1, SRSF2, and SRSF3 were inserted into the HindIII/EcoRI or EcoRI/BamHI (SRSF3) sites of p3XFLAG-CMV (Sigma-Aldrich).
3
Transient Expression of SARS-CoV PLpro in A549 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human alveolar basal epithelial A549 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; HyClone Laboratories, Logan, Utah, USA) with 100 U/mL of penicillin and streptomycin, 2 mM L-glutamine, and 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel). SARS-CoV PLpro gene, nt 4507–5840 of the SARS-CoV TW1 strain (GenBank accession no. AY291451 ) was amplified by RT-PCR, and then cloned into expression vector pcDNA3.1/His C (Invitrogen), as described in our prior reports12 (link)13 (link). The empty vector pcDNA3.1 or pSARS-PLpro at the concentrations of 0, 0.5, 1, 2, 5, and 10 μg/ml was transfected into A549 cells with Arrest-In transfection reagent (Thermo scientific). After 5-h incubation, transfected cells were maintained in DMEM medium containing 20% FBS. Transient expression of recombinant PLpro in A549 cells 2 days post transfection was analyzed using immunefluoresce staining and Western blotting with mouse polyclonal antibodies against anti-E. coli synthesized PLpro, as described in our prior reports12 (link)13 (link).
4
Modulation of miR-200 and ZEB1 in cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pLenti4.1-miR-200 was obtained from Dr. Gregory Goodall (University of Adelaide) and used as described elsewhere2 (link),7 (link),28 (link). A modified doxycycline inducible pTRIPz-RFP vector expressing miR-200a, miR-200b, or in combination (miR-200ab) was generously provided to us by Dr. Gregory Goodall at the University of Adelaide. Mouse ZEB1 was subcloned into pcDNA3.1/His C (Invitrogen) to generate the ZEB1 overexpression vector. shRNA to mouse ZEB1 and PD-L1 and scrambled controls in the pGFP-V-RS vector were obtained from OriGene. Mouse PD-L1 expression vector pUNO1-mCD274 was purchased from Invivogen. Pre-microRNAs miR-200c and negative control (scrambled oligos) (Life Technologies) were reverse transfected into cells with Lipofectamine 2000 (Life Technologies) at a final concentration of 50 nM.
5
SARS-CoV PLpro Expression and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human alveolar basal epithelial A549 cells grew in Dulbecco’s Modified Eagle’s Medium (HyClone Laboratories) and were transfected with control vector pcDNA3.1/His C (Invitrogen), or pSARS-PLpro containing SARS-CoV PLpro gene, as described in our prior reports (Li et al., 2011 (link), Li et al., 2012 (link), Li et al., 2016a , Li et al., 2016b (link)). In addition, pSARS-PLpro(H273A) that had the alanine substitution for histidine at position 273 by Ala within PLpro gene was constructed using PCR-based site-directed mutagenesis with a mutated primer pair (5′-GGTAACT ATCAGTGTGGTGCTTACACTCATATAACTGCTAAG-3′ and 5′-CTTAGCAGT TATATGAGTGTAAGCACCACACTGATAGTTACC-3′). A549 cells transiently expressing recombinant PLpro 2 days post transfection was analyzed using Western blotting, real-time RT-PCR, Sirius staining, and immunofluorescent staining assays.
6
SARS-CoV Genome Fragments Cloning
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The nucleotide sequences of SARS-CoV full-length SUD, SUD-NM, and SUD-MC within the SARS-CoV genome nucleotides 3887–4941, 3887–4673, and 4311–4880 (GenBank accession no. AY291451), respectively, were amplified using PCR with specific primer pairs. SARS-CoV replicon [36 (link)] kindly provided by Zhengli Shi (Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China) was used as the template. The primer pairs are shown in Table 1 and used for cloning the full-length SUD, SUD-NM, SUD-MC, SUD-N, SUD-M and SUD-C subdomains, respectively. The PCR products with a restriction site KpnI at 5′ end and a restriction site XhoI at 3′ end were cloned into the expression vector pcDNA3.1/His C (Invitrogen, Carlsbad, CA, USA) after double digest reaction with KpnI and XhoI, and then the resultant recombinant plasmids were named as pSUD-FL, pSUD-NM, pSUD-MC, pSUD-N, pSUD-M, and pSUD-C, respectively. In addition, firefly luciferase reporter plasmids IP-10GL3, tIP-10GL3, and IP-10mκB1, as gifted from David Proud at University of Calgary (Canada), contain wild type (−875/+97), truncated (−279/+97), and NF-κB1 site mutant CXCL10 promoter, respectively.
7
Desmin Mutant Transfection in C2C12 Cells
8
Modulation of miR-200 and ZEB1 in cancer
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!