Antibodies used in this study are listed in Table S1 . The phospho-CENPA (Ser18) antibody was generated and provided as a kind gift by Cell Signaling Technology. HA-tagged WT or mutant FBW7 and Cyclin E1 were described previously (24 (link)). HA-CCNE1 R130A mutant was generated by site-directed mutagenesis and confirmed by sequencing. Full length HA tagged CENP-A was amplified by PCR with a 5′ primer that introduced an BamHI site and an HA tag and a 3′ primer that introduced an SalI site. The PCR product was digested with BamHI and SalI and cloned into pLenti CMV GFP vector (Addgene). Mutant CENP-A constructs were generated by site-directed mutagenesis and confirmed by sequencing.
Plenti cmv gfp vector
Manufactured by Addgene
Sourced in United States
The PLenti-CMV-GFP vector is a lentiviral expression vector designed for the expression of the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. This vector can be used for the stable transduction and expression of GFP in a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Plko 1 puro vector, by Addgene (1 mentions)
Pgl3 basic luciferase vector, by Promega (1 mentions)
Pgpu6 gfp neo vector, by GenePharma (1 mentions)
Ha ub, by GenePharma (1 mentions)
Glucose, by Thermo Fisher Scientific (1 mentions)
Hcclm3, by Merck Group (1 mentions)
Hepg2, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Polybrene, by Merck Group (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Hepg2, by National Collection of Authenticated Cell Cultures (1 mentions)
Mhcc97h, by American Type Culture Collection (1 mentions)
Hcclm3, by American Type Culture Collection (1 mentions)
Hl 7702, by National Collection of Authenticated Cell Cultures (1 mentions)
Bel 7402, by National Collection of Authenticated Cell Cultures (1 mentions)
Sk hep1, by National Collection of Authenticated Cell Cultures (1 mentions)
4 protocols using plenti cmv gfp vector
1
Characterization of CENPA Mutants
2
Lentiviral Knockdown and Overexpression of DDX18
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For DDX18 knockdown, two different DDX18 shRNA target sequences were synthesized and inserted into pLKO.1-puro vector (Plasmid #8453, Addgene, Watertown, MA, USA). To overexpress DDX18, full-length DDX18 cDNA was inserted into pLenti-CMV GFP vector (Plasmid #17448, Addgene) linearized by BamHl/Sall digestion. For lentivirus production, package plasmid psPAX2 (Plasmid #12260, Addgene), envelop plasmid pMD2.G (Plasmid #12259, Addgene), and vectors were co-transfected into HEK 293FT cells. Then, 72 h after transfection, the supernatant was harvested and concentrated with Lenti-X concentrator (ECOTOP, Guangzhou, China). One day before the viral infection, cells were plated into a 6-well plate at a density of 1 × 105 cells/well and transduced in the presence of 5 μg/mL polybrene for 24 h. The cells were selected for puromycin resistance (1.5 μg/mL) for 48 h.
3
Cloning and Modulation of SPOP in FHC Cells
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The full-length ORF of SPOP (1125 bp, NM_001007226.1) was amplified from cDNA of FHC cells. The primers were as follows: F: 5′-AGAGAATTCATGTCAAGGGAAATCTTTGC-3′, R: 5′-AGAGGATCCTTAGGATTGCTTCAGGCGTT-3′. To construct the lentivirus production containing SPOP, the ORF of SPOP was subcloned into the pLenti-CMV-GFP vector (Addgene, Cambridge, MA, USA).
The synthesized DNA fragments encoding the short-hairpin RNA (shRNA) used for the knockdown of endogenous SPOP were inserted into the pGPU6/GFP/Neo vector (GenePharma, Shanghai, China). The sequences of the shRNAs were as follows: shSPOP, 5′-AACGCCTGAAGCAATCCTACTCGAGTAGGATTGCTTCAGGCGTT-3′, NC, 5′-TTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3′. All plasmids were verified by sequencing.
Serial deletion fragments of SPOP promoter region (−1320, −1167, −655, −350, −221, −189, −10/+472) were amplified by PCR and cloned into a pGL3-basic luciferase vector (Promega). A NheI site was added to the 5′-primer, and a XhoI site was added to the 3′-primer. The constructs were verified by sequencing of both DNA strands. The sequences of mutated sites were synthesized by Genescript (Nanjing, China).
FLAG-Gli2, HA-Ub and pcDNA3.1(-)A-RXRA were purchased from GenePharma.
The synthesized DNA fragments encoding the short-hairpin RNA (shRNA) used for the knockdown of endogenous SPOP were inserted into the pGPU6/GFP/Neo vector (GenePharma, Shanghai, China). The sequences of the shRNAs were as follows: shSPOP, 5′-AACGCCTGAAGCAATCCTACTCGAGTAGGATTGCTTCAGGCGTT-3′, NC, 5′-TTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3′. All plasmids were verified by sequencing.
Serial deletion fragments of SPOP promoter region (−1320, −1167, −655, −350, −221, −189, −10/+472) were amplified by PCR and cloned into a pGL3-basic luciferase vector (Promega). A NheI site was added to the 5′-primer, and a XhoI site was added to the 3′-primer. The constructs were verified by sequencing of both DNA strands. The sequences of mutated sites were synthesized by Genescript (Nanjing, China).
FLAG-Gli2, HA-Ub and pcDNA3.1(-)A-RXRA were purchased from GenePharma.
4
Cell Line Authentication and Transduction
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HL7701, HL-7702, HepG2, Huh7, Sk-Hep1, Bel7402, HLE, HLF and Alex cell lines were purchased from China Center for Type Culture Collection (CCTCC, Wuhan, China). MHCC-97 H and HCC-LM3 cell lines were obtained from Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China. 293T cells were purchased from the American Type Culture Collection. Cells were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 4.5 g/L glucose and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). All cell lines have been tested for their authenticity. Transfection were performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacture’s instruction. For lentivirus production, pLKO.1 vector and pLKO.1-shSHC4 or pLenti-CMV-GFP vector and pLenti-CMV-GFP-SHC4 (6 µg), psPAX2 (4.5 µg), pMD2.G (1.5 µg) were purchased from Addgene, Inc. (Cambridge, MA, USA) and were co-transfected into 293T cells. The virus-containing supernatants were collected and filtered 48 h after transfection. Freshly made virus supernatants supplemented with 8 µg/mL polybrene (Sigma-Aldrich) were added to exponentially growing HepG2, Huh7 or HCC-LM3 cells. After 8 h, fresh medium was added. SHC4 stable overexpression or knockdown cells were achieved by 1-week puromycin (5 µg/mL, Ann Arbor, MI, USA) selection.
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