Figure 7.

Loss of HECT domain leads to stabilization of U5 spliceosome. (A) Western blot of proteins identified in MS in splenic B cells (B220⁺) Mb1^WT/CRE;Ubr5^WT, Mb1^WT/CRE;Ubr5^ΔHECT/WT, and Mb1^WT/CRE;Ubr5^ΔHECT mice. (B) Western blot of UBR5 and spliceosome components in follicular B cells (B220⁺CD21⁺CD23⁺) Ubr5^WT and Ubr5^ΔHECT after treatment with cycloheximide with a table of average half-lives of UBR5 and spliceosome proteins. (C) 10% to 30% glycerol fractionation of nuclear lysate from HEK293T cells followed by western blot for UBR5 and spliceosome components. (D) Western blot of UBR5 and spliceosome components in follicular B cells (B220⁺CD21⁺CD23⁺) of Ubr5^WT and Ubr5^ΔHECT following MG132 treatment. (E) Western blot of UBR5 and known UBR5 substrate, RNF168, in follicular B cells (B220⁺CD21⁺CD23⁺) of Ubr5^WT and Ubr5^ΔHECT with or without irradiation exposure. (F) Half-life of RNF168 with cycloheximide treatment in follicular B cells (B220⁺CD21⁺CD23⁺) before and after irradiation exposure. (G) Western blot of spliceosome components in pro (B220⁺IgM⁻ckit⁺), pre (B220⁺IgM⁻CD25⁺), and immature (B220⁺IgM^loIgD⁻) B-cell populations from the BM and marginal zone (B220⁺CD21⁺CD23⁻), and follicular (B220⁺CD21⁺CD23⁺) B-cell populations from the spleen. (H) RT-PCR of mRNA in Mb1^WT/CRE;Ubr5^WT and Mb1^WT/CRE;Ubr5^ΔHECT follicular B cells (B220⁺CD21⁺CD23⁺).