A widely used pan-isoform-FBXW7 antibody used in cell cycle studies does not detect FBXW7

Hannah Ennis, Denise M McDonald*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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Abstract

FBXW7 is the substrate recognition component of the E3 ubiquitin ligase SCFFBW7 complex which controls the levels of CYCLINE, c-MYC and HIF1α proteins crucial for cell growth and differentiation. Mutations in FBXW7 are frequently associated with tumourigenesis. While examining FBXW7 regulation we were compelled to reevaluate a commonly used anti-FBXW7 antibody. Retinal microvascular endothelial cells (RMEC) were exposed to normoxia (21% oxygen) or hypoxia (1% oxygen) for 24 h or treated with MG132 and protein extracted for western blotting. Flag-tagged FBXW7-α, β or γ isoforms were transfected into HEK293A cells and processed using denaturing and native extraction protocols for western blotting or immunoprecipitation analysis. Two anti-FBXW7 antibodies were used, one raised to the unique FBXW7α N-terminus and the other to the C-terminus region common to all isoforms. Initial studies showed that the pan-isoform C-terminus antibody detected a single 64kDa band in RMEC rather than any of the predicted sizes for FBXW7. In contrast, expression of the isoform-specific constructs, detected with an anti-Flag antibody, confirmed the expected migratory distance of 110kDa, 68kDa and 65kDa for α, β and γ respectfully. Similarly, the N-terminus FBXW7α antibody also detected the 110kDa product. Notably, the C-terminus antibody did not recognize any of the isoforms but continued to detect a 64kDa band in all samples, including the non-transfected controls. Immunoprecipitation confirmed this lack of specificity and the inability to detect overexpressed or endogenous FBXW7α in HEK293A cells and RMEC. A commonly used C-terminus FBXW7 antibody does not detect FBXW7 under standard western blotting conditions.

Original languageEnglish
Number of pages11
JournalCell Cycle
Early online date14 May 2023
DOIs
Publication statusEarly online date - 14 May 2023

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