C myc

Manufactured by New England Biolabs

Sourced in Germany, United States

The c-Myc antibody is a primary antibody that specifically recognizes the c-Myc protein, a regulator of cell growth and proliferation. It can be used for applications such as Western blotting, immunoprecipitation, and immunohistochemistry.

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Lab products found in correlation

Dual glo luciferase, by Promega (1 mentions) Lipofectamine 2000, by OriGene (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) β actin, by Merck Group (1 mentions) Prism software, by GraphPad (1 mentions)

3 protocols using c myc

c-MYC Transcriptional Reporter Assay

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HEK293T cells were seeded at 35,000 cells/well in a 96-well plate. Cells were transfected with the MYC luciferase reporter according to protocol described above using Lipofectamine 2000 (Invitrogen, 11668019) as a transfection reagent. Cells were also transfected with either FLAG-eGFP, FLAG-tagged wild-type c-MYC (Origene, RC201611) or C171Y cMYC mammalian expression plasmids using Lipofectamine 2000. Cells were transfected with 1 μg/well of corresponding plasmid in a final ratio of 3:1 Lipofectamine 2000:DNA. The c-MYC C171Y mutant plasmid was generated using Q5 site-directed mutagenesis kit according to the manufacturer’s instructions (New England Biolabs, E0554S). Sequencing was confirmed with Quintara Biosciences. 24 hours post-transfection, media was carefully aspirated from each well, and 50 μL of fresh media containing either DMSO or compound was added. After 24 hours of compound treatment, Dual-Glo luciferase (Promega, E2920) detection was performed according to manufacturer’s protocol. Firefly and Renilla luminescence were read on a SpectraMax i3 plate reader. Background luminescence was subtracted using a blank control then Firefly:Renilla was calculated for each individual well. Lysates were also obtained for confirmation FLAG-protein overexpression by Western blotting.

Boike L., Cioffi A.G., Majewski F.C., Co J., Henning N.J., Jones M.D., Liu G., McKenna J.M., Tallarico J.A., Schirle M, & Nomura D.K. (2020). Discovery of a Functional Covalent Ligand Targeting an Intrinsically Disordered Cysteine Within MYC. Cell chemical biology, 28(1), 4-13.e17.

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Quantitative Western Blot Analysis

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To evaluate (phosphorylated) protein expression of LDH-A, STAT-3/pSTAT-3, and c-myc, whole cell lysates were prepared with RIPA-buffer and samples (40 μg) were subjected to Western blotting (10% SDS-PAGE). Membranes were sequentially analyzed with antibodies to GAPDH2 (Santa Cruz Biotechnology, Heidelberg, Germany), c-myc, LDH-A, STAT-3, phosphorylated STAT-3 (pSTAT-3) (all Cell Signaling, New England Biolabs GmbH, Germany), and β-actin (Sigma-Aldrich, Germany) in dry milk (1%). Expression was measured by chemo-luminescence (ECL Western Blot Bright, Biozym, Germany). Intensities of protein bands were measured with ImageJ software and protein regulation of 3 Western blots each (n = 3) was calculated by normalization to loading and treatment control using GraphPad Prism software (version 6, GraphPad Software, USA).

Leidgens V., Seliger C., Jachnik B., Welz T., Leukel P., Vollmann-Zwerenz A., Bogdahn U., Kreutz M., Grauer O.M, & Hau P. (2015). Ibuprofen and Diclofenac Restrict Migration and Proliferation of Human Glioma Cells by Distinct Molecular Mechanisms. PLoS ONE, 10(10), e0140613.

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Flow Cytometry Quantification of Oncoproteins

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Flow cytometry methodology has been previously described 5, 36 and was used for the detection of PP2Ac, PP2A Y307 , CIP2A, c-Myc, c-Myc S62 , E2F1 and E2F1 S364 . The following antibodies were used: Anti-PP2A catalytic subunit (Merck Millipore, UK), PP2A Y307 (Epitomics, USA), CIP2A, E2F1 and E2F1 S364 (Santa Cruz Biotechnology, USA), c-Myc (New England Biolabs, UK), c-Myc S62 (Abcam, UK), anti-mouse and anti-rabbit Alex fluor 488 (Invitrogen, UK). Levels of pCrKL and CrKL were used as an assay of BCR-ABL1 activity, measured by flow cytometry as previously described. 36 Supplementary information is available at Leukemia's website.

Lucas C.M., Harris R.J., Holcroft A.K., Scott L.J., Carmell N., McDonald E., Polydoros F, & Clark R.E. (2015). Second generation tyrosine kinase inhibitors prevent disease progression in high-risk (high CIP2A) chronic myeloid leukaemia patients. Leukemia, 29(7).

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