RPMI1640 medium, Opti-modified Eagle’s medium (Opti-MEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Life Technologies, Inc. (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO), RNase A, leupeptin, aprotinin, phenylmethylsulfonylfluoride, and Triton X-100 were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The pCMV-Neo-Bam p53-R175H, pCMV-Neo-Bam p53-R248W, pCMV-Neo-Bam p53-R273H, and pCMV-Neo-Bam p53 wild-type were obtained from Addgene (Cambridge, MA, USA). The antibodies against anti-p53, anti-Rad21, anti-S1PR1, anti-THBS1, and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The protein G PLUS-agarose was also obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Pcmv neo bam p53 r248w
Manufactured by Addgene
Sourced in United States
PCMV-Neo-Bam p53 R248W is a plasmid vector. It contains the p53 gene with the R248W mutation.
Automatically generated - may contain errors
Lab products found in correlation
Pcmv neo bam p53 r273h, by Addgene (3 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Pcmv neo bam p53 r175h, by Addgene (2 mentions)
Pcmv neo bam p53 wild type, by Addgene (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions)
Aprotinin, by Merck Group (2 mentions)
Leupeptin, by Merck Group (2 mentions)
Anti s1pr1, by Santa Cruz Biotechnology (1 mentions)
Anti thbs1, by Santa Cruz Biotechnology (1 mentions)
Opti modified eagle s medium opti mem, by Thermo Fisher Scientific (1 mentions)
Protein g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Rnase a, by Merck Group (1 mentions)
Anti p53, by Santa Cruz Biotechnology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Mv4 11, by Lonza (1 mentions)
Aml 193, by American Type Culture Collection (1 mentions)
Amaxa nucleofector, by Lonza (1 mentions)
5 protocols using pcmv neo bam p53 r248w
1
Characterization of Mutant p53 Proteins
2
Generation and Validation of Mutant p53 MV4;11 Cells
3
Plasmid Construction and Genomic Targeting
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A plasmid bearing the p53 R248W mutation (pCMV-Neo-Bam p53 R248W) was purchased from Addgene (Cambridge, MA, USA). The open reading frame was cloned into the pEF vector97 (link) along with a FLAG epitope. The pEF-FLAG-MBP plasmid was previously described43 (link). The following oligonucleotide pairs were annealed and ligated into BsmB1-cut pFgh1tUTG98 (link).
ATM exon 2: 5′-TCCCTTGTTTCAGGATCTCGAATC-3′, 5′-AAACGATTCGAGATCCTGAAACAA-3′
TP53 exon 1: 5′-TCCCTCGACGCTAGGATCTGACTG-3′, 5′-AAACCAGTCAGATCCTAGCGTCGA-3′
PRKDC exon 1: 5′-TCCCGCCGGTCATCAACTGATCCG-3′, 5′-AAACCGGATCAGTTGATGACCGGC-3′
RAD51A exon 2: 5′-TCCCTAGCTCCTTCTTTGGCGCAT-3′, 5′-AAACATGCGCCAAAGAAGGAGCTA-3′
ATM exon 2: 5′-TCCCTTGTTTCAGGATCTCGAATC-3′, 5′-AAACGATTCGAGATCCTGAAACAA-3′
TP53 exon 1: 5′-TCCCTCGACGCTAGGATCTGACTG-3′, 5′-AAACCAGTCAGATCCTAGCGTCGA-3′
PRKDC exon 1: 5′-TCCCGCCGGTCATCAACTGATCCG-3′, 5′-AAACCGGATCAGTTGATGACCGGC-3′
RAD51A exon 2: 5′-TCCCTAGCTCCTTCTTTGGCGCAT-3′, 5′-AAACATGCGCCAAAGAAGGAGCTA-3′
4
Assessing p53 mutant protein expression
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Expression studies were approved by the Keele genetic modifications of microorganisms committee. SkOv-3 cells (32,000 cells per well of a 24 well plate) were transfected with 0.1 μg CMV-Neo-Bam (vector), pCMV-Neo-Bam p53 wt, pCMV-Neo-Bam p53 R175H, pCMV-Neo-Bam p53 R273H, pCMV-Neo-Bam p53 R248W (Addgene) and 0.2 μL of Lipofectamine 2000 as previously described47 (link). Protein expression was measured by immunoblotting.
5
Integrin β4-Mediated Signaling in Cancer Cells
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RPMI 1640 medium, foetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO), RNase A, leupeptin, aprotinin, phenylmethylsulfonylfluoride, selective FAK inhibitor 14 (1,2,4,5-benzenetetramine tetrahydrochloride), and Triton X-100 were purchased from Sigma–Aldrich Co. (St Louis, MO, USA). The pCMV-Neo-Bam p53 R248W, pCMV-Neo-Bam p53 R273H, and pCMV-Neo-Bam p53 wild-type were obtained from Addgene (Cambridge, MA). CellTrackerTM was obtained from Invitrogen (Grand Island, NY, USA). The antibodies anti-p53 (DO-1, sc-126), anti-Akt, anti-focal adhesion kinase (FAK), integrin β4, and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies anti-phospho-Akt and anti-phospho-FAK were purchased from Cell Signalling Technology (Beverly, MA, USA). Function-blocking antibody against the human integrin β4 was obtained from EMD Millipore (Billerica, MA, USA)53 (link). The PI3K/Akt inhibitor LY294002 and wortmannin were obtained from Calbiochem (San Diego, CA, USA).
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