Protocol detail

Myc and Fbw7 Mutational Analysis

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Myc and all mutants thereof are in pCS2+ vector containing a single HA tag at the N terminus. Fbw7 and all mutants thereof are in p3xFLAG-CMV24 (Sigma-Aldrich), except HA-Fbw7, which was cloned into pCS2+ with a single N-terminal HA tag. All mutations were generated by the QuikChange method (Stratagene) and verified by sequencing. Fbw7 ∆F has the entire F-box deleted, while Fbw7 ∆D has a deletion of five critical amino acids in the dimerization domain (14 (link)). The following antibodies were used: FLAG: M2 (Sigma-Aldrich), HA: Y11 (Santa Cruz Biotechnology), Myc: D3N8F (Cell Signaling Technology), pT58: EPR17923 (Abcam ab185655), pS62: ERP17924 (Abcam ab185656), pT244: in-house mouse monoclonal, Fbw7: A301-720A and A301-721A (Bethyl Laboratories), proliferating cell nuclear antigen (PCNA): PC-10 (Santa Cruz Biotechnology), and GSK3: 368662 (Calbiochem).

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Welcker M., Wang B., Rusnac D.V., Hussaini Y., Swanger J., Zheng N, & Clurman B.E. (2022). Two diphosphorylated degrons control c-Myc degradation by the Fbw7 tumor suppressor. Science Advances, 8(4), eabl7872.

Publication 2022

p3xFLAG-CMV24 FLAG: M2 HA: Y11 D3N8F

A301 Amino acids Antibodies Deletion Dimerization Gsk3 House mouse Mutations Proliferating cell nuclear antigen Vector

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Variable analysis

independent variables

Myc and all mutants thereof
Fbw7 and all mutants thereof

dependent variables

Protein interactions between Myc, Fbw7, and their mutants
Phosphorylation of Myc at T58, S62, and T244

control variables

HA tag at the N terminus of Myc and HA-Fbw7
All mutations were generated by the QuikChange method (Stratagene) and verified by sequencing
Fbw7 ∆F has the entire F-box deleted
Fbw7 ∆D has a deletion of five critical amino acids in the dimerization domain

positive controls

FLAG: M2 (Sigma-Aldrich)
HA: Y11 (Santa Cruz Biotechnology)
Myc: D3N8F (Cell Signaling Technology)
Fbw7: A301-720A and A301-721A (Bethyl Laboratories)
PCNA: PC-10 (Santa Cruz Biotechnology)
GSK3: 368662 (Calbiochem)

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