Full-length human GALNT1 from non-tumorous liver was cloned by using RT-PCR with sense primer 5′-CCCAAGCTTGGGCCATGAGA AAATTTGCATA-3′ and antisense primer 5′-CGGGATCCCTCAGA ATATTTCTGGCAGGG-3′. The RT-PCR products were cloned into pcDNA3.1A (Invitrogen, Life Technologies) to generate pcDNA3.1A/GALNT1 gene and confirmed by DNA sequencing.
Pcdna3.1a
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PcDNA3.1A is a mammalian expression vector designed for high-level, transient expression of recombinant proteins in a wide range of cell lines. It features a strong cytomegalovirus (CMV) promoter, a multiple cloning site, and a polyadenylation signal to facilitate efficient transcription and translation of the target gene.
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Lab products found in correlation
Puromycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Pgex 4t vector, by GE Healthcare (1 mentions)
Quikchange multi site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Herculase 2 fusion, by Agilent Technologies (1 mentions)
Btx square wave electroporator, by Harvard Apparatus (1 mentions)
Pact2, by Takara Bio (1 mentions)
Pgex 4t 1, by Takara Bio (1 mentions)
Human fetal brain cdna library, by Takara Bio (1 mentions)
8 protocols using pcdna3.1a
1
Cloning of Full-length Human GALNT1
2
Establishment of hSGLT1-Expressing CHO Cell Line
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Chinese hamster ovary-K1 (CHO-K1) cells were cultured in Ham’s F-12 medium supplemented with 10% FCS and penicillin–streptomycin. A human SGLT1 expression vector (pcDNA3.1-SGLT1) was constructed as follows: the open reading frame of human SGLT1 was amplified from a plasmid encoding human SGLT1 (Flexi clone, Promega) using the primer 5’-GGGAAGCTTATGGACAGTAGCACCTGGAG-3’, which introduced a HindIII site, and the primer 5’-GGGGAATTCGGCAAAATATGCATGGCAAAAG-3’, which introduced an EcoRI site. The HindIII/EcoRI-digested PCR fragment was ligated into the digested pcDNA3.1A (Invitrogen, Carlsbad, CA, USA), thereby creating pcDNA-hSGLT1, and sequenced.
The plasmid vector was transfected into CHO-K1 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. hSGLT1-expressing cells (transfected cells) were selected with G418, and limiting dilution was performed in 96-well plates. Several single clones were selected from the 96-well plates. The clone that exhibited the strongest sodium-dependent [3H]-glucose uptake activity was selected and the hSGLT1 stable cell line was established.
The plasmid vector was transfected into CHO-K1 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. hSGLT1-expressing cells (transfected cells) were selected with G418, and limiting dilution was performed in 96-well plates. Several single clones were selected from the 96-well plates. The clone that exhibited the strongest sodium-dependent [3H]-glucose uptake activity was selected and the hSGLT1 stable cell line was established.
3
Stable XBP1 Knockdown and Overexpression
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For stable knockdown of XBP1, siRNA sequences Sense: 5′-AAAGACAGCAAGTGGTAGATTTA-3′; Anti-sense: 5′-AAAATAAATCTACCACTTGCTGT-3′52 (link) was cloned into pFIV-H1/U6 vector (System Biosciences), while a non-targeting sequence was cloned as the negative control vector. The resulting siRNA vector construct or pFIV-H1/U6-copGFP vector (System Biosystems), which contains CopepodGFP coding sequence in replacement of the puromycin selection marker, was co-transfected with lentiviral packaging vectors pFIV-34N and pVSV-G into HEK-293T cells with TurboFect transfection reagent (Thermo Scientific). Culture supernatant was collected after 48 h and used to resuspend U937 cells, with the addition of polybrene to a final concentration of 8 μg/mL. The resulting suspensions were then centrifuged at 1000 g for 2 h at 37°C, after which the transduced cells were re-suspended in 2 mL of fresh complete RPMI media and seeded into 6 well plates. Successfully transduced cells were selected with 1 μg/mL puromycin (Life technologies) in culture medium 48 h after transduction, for 1.5 weeks.
For XBP1s overexpression, XBP1s coding sequence (NM_001079539.1) was cloned into pcDNA3.1A (Invitrogen) and transfected into A549 cells using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer's instructions.
For XBP1s overexpression, XBP1s coding sequence (NM_001079539.1) was cloned into pcDNA3.1A (Invitrogen) and transfected into A549 cells using X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer's instructions.
4
Plasmid construction and mutagenesis methods
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The plasmids encoding Arl1 and arfaptin-1 were constructed using PCR. Using a two-step PCR procedure, we replaced the codon for Gln71 with the codon for Leu to generate a construct encoding Arl1QL. To generate the glutathione-S-transferase (GST) fusion construct for expression in E. coli, we subcloned the fragment encoding Arl1QL into the pGEX4T vector (GE Healthcare) via the EcoRI site. The plasmids encoding siRNA-resistant arfaptin-1a (arfaptin-1am) and siRNA-resistant arfaptin-1b (arfaptin-1bm) were generated using the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Stratagene Products Division, La Jolla, CA, USA), according to the manufacturer’s instructions. The plasmids encoding arfaptin-1a-S100A, arfaptin-1a-S100D, arfaptin-1b-S132A, arfaptin-1b-S132D and arfaptin-1b-F317A were generated using a two-step PCR procedure. To generate the myc-tagged construct for expression in HeLa cells, full length arfaptin-1a and arfaptin-1b, siRNA-resistant arfaptin-1a and arfaptin-1b, truncated arfaptin-1b, and mutated arfaptin-1b were subcloned into pcDNA3.1A (Invitrogen). The sequences encoding golgin-97-GRIP and golgin-245-GRIP were amplified by PCR and subcloned into pEGFP-C2 (Clontech Laboratories, Inc., Mountain View, CA USA). The sequences of all of the primers used for plasmid construction are shown in S2 Table .
5
Generation of FGFR3 Constructs
6
Granzyme B-Induced Apoptosis Assay
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1x107 Karpas 299 cells were transfected with 50 μg of GzB cDNA in pcDNA 3.1A (Invitrogen) or 50 μg of pcDNA 3.1A alone as previously described [64 (link)] using a BTX square wave electroporator (Harvard Apparatus; Holliston MA). 48 h post-transfection, cells were treated with staurosporine and apoptosis was analysed by TUNEL as described above.
7
Generating Stable Cell Lines with NF90 Manipulation
8
Cloning and Expressing ARL4 Proteins
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cDNA sequences of Arl4A/C/D and its mutants were cloned as previously described (Li et al., 2007 (link)). Briefly, Arl4A was cloned into the mammalian expression vector pSG5 (Stratagene, La Jolla, CA) to generate untagged Arl4A. The cDNA sequence was subcloned into pET32a (Novagen, Madison, WI) for His-tagged Arl4A expressed in Escherichia coli or pBTM116 for LexA-tagged Arl4A expressed in yeast. Full-length Robo1 and srGAP1 cDNA sequences were amplified from a human fetal brain cDNA library (Clontech) using PCR. Robo1 and srGAP1 were cloned into the mammalian expression vectors pCMV-Tag4A and pcDNA3.1A (Invitrogen), the E. coli expression vector pGEX4T-1, or the yeast expression vector pACT2 (Clontech). Replacement of alanine was accomplished using a two-step PCR technique. All constructs were confirmed by DNA sequencing.
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