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A Model diagram of M-CSFR structure and its potential <t>ubiquitination</t> site. B Highly conserved lysine (K) residues (K791) on M-CSFR from different species. C Western blot analysis of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or its lysine mutants) and Myc-Cbl-b and then treated with M-CSF (50 ng/mL) and CHX (50 µM) for 2 h. D Western blot analysis of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or its lysine mutants) and Myc-c-Cbl and then treated with M-CSF (50 ng/mL) and CHX (50 µM) for 2 h. E Immunoprecipitation analysis of polyubiquitination of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or K791R), HA-Ub and Myc-Cbl-b and then treated with M-CSF (50 ng/mL) for 2 h. F Immunoprecipitation analysis of polyubiquitination of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or K791R), HA-Ub and Myc-c-Cbl and then treated with M-CSF (50 ng/mL) for 2 h. G Immunoprecipitation analysis of polyubiquitination types of M-CSFR in HEK293T cells cotransfected with His-M-CSFR, Myc-Cbl-b and different types of HA-Ub and then treated with M-CSF (50 ng/mL) for 2 h. H Immunoprecipitation analysis of polyubiquitination types of M-CSFR in HEK293T cells cotransfected with His-M-CSFR, Myc-c-Cbl and different types of HA-Ub and then treated with M-CSF (50 ng/mL) for 2 h. I WT and dKO BMDMs were starved of M-CSF for 24 h and restimulated with M-CSF (50 ng/mL) for 3 min and harvested for Co-immunoprecipitation analysis of K63-linked polyubiquitination of M-CSFR.
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A Model diagram of M-CSFR structure and its potential ubiquitination site. B Highly conserved lysine (K) residues (K791) on M-CSFR from different species. C Western blot analysis of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or its lysine mutants) and Myc-Cbl-b and then treated with M-CSF (50 ng/mL) and CHX (50 µM) for 2 h. D Western blot analysis of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or its lysine mutants) and Myc-c-Cbl and then treated with M-CSF (50 ng/mL) and CHX (50 µM) for 2 h. E Immunoprecipitation analysis of polyubiquitination of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or K791R), HA-Ub and Myc-Cbl-b and then treated with M-CSF (50 ng/mL) for 2 h. F Immunoprecipitation analysis of polyubiquitination of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or K791R), HA-Ub and Myc-c-Cbl and then treated with M-CSF (50 ng/mL) for 2 h. G Immunoprecipitation analysis of polyubiquitination types of M-CSFR in HEK293T cells cotransfected with His-M-CSFR, Myc-Cbl-b and different types of HA-Ub and then treated with M-CSF (50 ng/mL) for 2 h. H Immunoprecipitation analysis of polyubiquitination types of M-CSFR in HEK293T cells cotransfected with His-M-CSFR, Myc-c-Cbl and different types of HA-Ub and then treated with M-CSF (50 ng/mL) for 2 h. I WT and dKO BMDMs were starved of M-CSF for 24 h and restimulated with M-CSF (50 ng/mL) for 3 min and harvested for Co-immunoprecipitation analysis of K63-linked polyubiquitination of M-CSFR.

Journal: Cell Death & Disease

Article Title: E3 Ubiquitin ligases Cbl-b and c-Cbl maintain the homeostasis of macrophages by regulating the M-CSF/M-CSFR signaling axis

doi: 10.1038/s41419-025-08047-4

Figure Lengend Snippet: A Model diagram of M-CSFR structure and its potential ubiquitination site. B Highly conserved lysine (K) residues (K791) on M-CSFR from different species. C Western blot analysis of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or its lysine mutants) and Myc-Cbl-b and then treated with M-CSF (50 ng/mL) and CHX (50 µM) for 2 h. D Western blot analysis of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or its lysine mutants) and Myc-c-Cbl and then treated with M-CSF (50 ng/mL) and CHX (50 µM) for 2 h. E Immunoprecipitation analysis of polyubiquitination of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or K791R), HA-Ub and Myc-Cbl-b and then treated with M-CSF (50 ng/mL) for 2 h. F Immunoprecipitation analysis of polyubiquitination of M-CSFR in HEK293T cells cotransfected with His-M-CSFR (WT or K791R), HA-Ub and Myc-c-Cbl and then treated with M-CSF (50 ng/mL) for 2 h. G Immunoprecipitation analysis of polyubiquitination types of M-CSFR in HEK293T cells cotransfected with His-M-CSFR, Myc-Cbl-b and different types of HA-Ub and then treated with M-CSF (50 ng/mL) for 2 h. H Immunoprecipitation analysis of polyubiquitination types of M-CSFR in HEK293T cells cotransfected with His-M-CSFR, Myc-c-Cbl and different types of HA-Ub and then treated with M-CSF (50 ng/mL) for 2 h. I WT and dKO BMDMs were starved of M-CSF for 24 h and restimulated with M-CSF (50 ng/mL) for 3 min and harvested for Co-immunoprecipitation analysis of K63-linked polyubiquitination of M-CSFR.

Article Snippet: When protein ubiquitination was examined, 10 mM N-ethylmaleimide (MedChemExpress, USA) was added into the NP-40 lysis buffer.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation