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. 2024 Jun 24;15(6):446. doi: 10.1038/s41419-024-06844-x

Fig. 5. VHL ubiquitylates UBE3B at lysine residues 286 and 427.

Fig. 5

A, B Ubiquitination of endogenous UBE3B in MDA-MB-231 cells (A) and T47D cells (B) with ectopic expression of WT or Y112H Flag-VHL. C, D Ubiquitination of endogenous UBE3B in control or VHL knockdown MDA-MB-231 cells (C) and T47D cells (D). E In vitro ubiquitination of His-UBE3B by GST-VHL. F HEK293T cells were co-transfected with Myc-UBE3B, Flag-VHL, and HA-ubiquitin. Immunoprecipitated Myc-UBE3B was subjected to mass spectrometry analysis to recover potential ubiquitination sites. The identified ubiquitination sites were indicated with an asterisk. G Ubiquitination of immunoprecipitated WT or mutant Myc-UBE3B by Flag-VHL in HEK293T cells treated with 10 μM MG132 for 6 h before harvest. H Ubiquitination of immunoprecipitated WT or K286/427R mutant (DM) Myc-UBE3B by Flag-VHL in MDA-MB-231 cells treated with 10 μM MG132 for 6 h before harvest. I Protein levels of WT or DM Myc-UBE3B in MDA-MB-231 cells with or without Flag-VHL. WCL whole-cell lysate.