Fbxw7

Manufactured by Fortis Life Sciences

Sourced in United States

FBXW7 is a protein involved in the ubiquitin-proteasome system, which plays a role in the degradation of target proteins. It functions as a substrate recognition component of the SCF (Skp1-Cullin-F-box) E3 ubiquitin-protein ligase complex.

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3 protocols using fbxw7

Immunoblotting Analysis of Protein Interactions

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Cells were lysed in EBC buffer (50 mM Tris pH 8.0, 120 mM NaCl, 1 mM EDTA, 0.5% NP40) containing 1 mM phenylmethylsulfonyl fluoride, 20 U/ml aprotinin, 1 µM leupeptin, 1 mM dithiothreitol, 0.1 mM NaF, 0.1 mM sodium orthovanadate, and 10 mM β-glycerophosphate. Proteins were resolved by SDS-PAGE, transferred to the membrane, and immunoblotted with the indicated antibodies: Fbxl8 (Santa Cruz Cat# sc-390582), Fbxw7 (Bethyl Laboratories Cat# A301-720A), c-myc (Cell Signaling Cat# 9402S), Cyclin D3 (Cell Signaling DCS22), Actin (Sigma Cat# A5316), vinculin (Cell Signaling Cat# 4650S), Alpha Tubulin (Sell Signaling Cat# 2144S) Anti-Flag (Sigma Cat#F7425-.2 MG), and anti-V5 (Sigma Cat#V8137-.2 MG). Proteins of interest were detected with horseradish peroxidase-conjugated antimouse or rabbit antibodies, and signals were visualized with the ECL system (Perkin Elmer Cat# NEL105001EA).

Bajpai S., Jin H.R., Mucha B, & Diehl J.A. (2022). Ubiquitylation of unphosphorylated c-myc by novel E3 ligase SCFFbxl8. Cancer Biology & Therapy, 23(1), 348-357.

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Antibody Characterization for Bacterial Research

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Anti-Enolase and anti-PspA serum (S. Hammerschmidt, University of Greifswald, Germany); anti-BgaA serum (S. King, The Ohio State University, USA); and antibodies specific for His₆ (Invitrogen, MA1-21315), K48-Ub linkage (Millipore, 05-1307), K63-Ub linkage (Millipore, 14-6077-80), Ply (Santa Cruz Biotechnology, sc-80500), FBXW7 (Bethyl Laboratories, A301-721A), SKP1 (Invitrogen, MA5-15928), Cullin1 (Invitrogen, 71-8700), glyceraldehyde phosphate dehydrogenase (Millipore, MAB374), GSK3β [Cell Signaling Technology (CST), D5C5Z], phosphothreonine (CST, 9381S), IgA, Kappa from murine myeloma, clone TEPC-15 (Sigma-Aldrich, M1421), and PSMB7 (Invitrogen, PA5-111404) were procured. The following secondary antibodies were used: horseradish peroxidase (HRP)–tagged anti-rabbit (BioLegend, 406401), HRP-tagged anti-mouse (BioLegend 405306), anti-rabbit Alexa Fluor 488 (Invitrogen, A27206), anti-rabbit Alexa Fluor 555 (Invitrogen, A31572), biotin-conjugated anti-mouse immunoglobulin A (Life Technologies, M31115), anti-mouse Alexa Fluor (Invitrogen, A31570), anti-mouse Alexa Fluor 488 (Invitrogen, A21202), anti-goat Alexa Fluor 633 (Invitrogen, A21082), and FM4-64 (Thermo Fisher Scientific, T13320).

Apte S., Bhutda S., Ghosh S., Sharma K., Barton T.E., Dibyachintan S., Sahay O., Roy S., Sinha A.R., Adicherla H., Rakshit J., Tang S., Datey A., Santra S., Joseph J., Sasidharan S., Hammerschmidt S., Chakravortty D., Oggioni M.R., Santra M.K., Neill D.R, & Banerjee A. (2023). An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins. Science Advances, 9(12), eade1851.

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Immunoblotting and Immunoprecipitation Protocol

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The following antibodies were used: FLAG (Sigma, #F7425), KMT2D (Bethyl Laboratories, #A300-BL1185), UTX (Bethyl Laboratories, #A302–374A), ASH2L (Bethyl Laboratories, #A300–489A), WDR5 (Bethyl Laboratories, #A302–430A), DPY30 (Bethyl Laboratories, #A304–296A), NCOA6 (Bethyl Laboratories, #A300–411A), CUL1 (Invitrogen, #718700), FBXW7 (Bethyl Laboratories, #A301–720), C-MYC (Santa Crus, #sc-764), c-MYC-pT58 (Applied Biological Materials, #Y011034), HA (Biolegend, #901513), HA (Cell Signaling Technology, #3724), α-Tubulin (Santa Crus, #sc-8035), Skp1 (Santa Cruz, #sc-7163), CyclinE1 (Cell Signaling Technology, #4129), Cdk2 (Santa Cruz, #sc-6248), CyclinE (clone HE12), Vinculin (Santa Cruz, #sc-73614), ubiquitin K48 (EMD Millipore, 05–1307), Rabbit IgG-HRP (GE Healthcare, NA934V), mouse IgG-HRP (GE Healthcare, NA931V) and anti-FLAG M2 Affinity Gel (Sigma, #A2220).

Saffie R., Zhou N., Rolland D., Önder Ö., Basrur V., Campbell S., Wellen K.E., Elenitoba-Johnson K.S., Capell B.C, & Busino L. (2020). FBXW7 triggers degradation of KMT2D to favor growth of diffuse large B-cell lymphoma cells. Cancer research, 80(12), 2498-2511.

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