Fig. 7: Stabilization of MEF2B C-terminal isoform-switch mutants by dissociation from the CUL3/KLHL12 complex.

a Detection of MEF2B/CUL3/KLHL12 interaction by Flag (MEF2B) immunoprecipitation (IP) using nuclear extracts of SUDHL10 cells expressing Flag-HA-MEF2B-WT-isoA, -isoB or isoform-switch mutants. Lamin B1 was used as loading control. Quantifications were normalized to the immunoprecipitated MEF2B (HA). Fold changes were calculated relative to the MEF2B WT-isoA sample. b MEF2B IP followed by immunoblotting (IB) using nuclear extracts of isogenic OCI-Ly7 clones including controls (Neutral) and cells expressing MEF2B isoform-switch (P256fs and P273fs) mutants. IgG IP was used as negative control. Quantifications were normalized to the immunoprecipitated MEF2B. Fold changes were calculated relative to the neutral clones. Samples are the same as in Fig. 5e. c Ubiquitination assay upon Flag (MEF2B) IP followed by IB using total extracts of HEK293T cells co-transfected with expression vectors for Flag-HA-MEF2B-WT-isoA, -isoB or isoform-switch mutants (P243fs, L269fs, and P273fs), and V5-Ub, Myc-KLHL12 and Myc-CUL3. Total ubiquitin and K48 ubiquitin chain type were detected. The samples derive from the same experiment but different gels for Ub(K48), in the IP samples, and V5 (pan-Ub), in the input samples, were processed in parallel. d Relative MEF2B protein levels, as measured upon CHX treatment for 12 h in SUDHL10 cells expressing Flag-HA-MEF2B-WT-isoA (n = 6) or isoform-switch mutants (n = 3 each). Numbers (n) refer to independent experiments. The complete CHX pulse-chase experiment is reported in Supplementary Fig. 8d. Data shown as average fold change ± SD compared to MEF2B-WT-isoA (two-tailed unpaired T test). e MEF2B relative expression level, as measured upon CHX treatment for 12 h in isogenic OCI-Ly7 control (Neutral; n = 3) and MEF2B isoform-switch mutant (n = 3 each) clones. Complete CHX pulse-chase experiment in Supplementary Fig. 8e. Data shown as average fold change ± SD relative to neutral clones (two-tailed unpaired T test). The experiments in (a) and (c) were repeated three times with similar results. Source data are provided as a Source Data file.