Fig. 6: Stabilization of MEF2B C-terminal missense mutants by dissociation from the CUL3/KLHL12 complex. | Nature Communications

Fig. 6: Stabilization of MEF2B C-terminal missense mutants by dissociation from the CUL3/KLHL12 complex.

From: MEF2B C-terminal mutations enhance transcriptional activity and stability to drive B cell lymphomagenesis

Fig. 6

a Detection of MEF2B/CUL3/KLHL12 interaction by MEF2B immunoprecipitation (IP) followed by immunoblotting (IB) from SUDHL10 and normal GC B cells. Lamin B1, nuclear fraction control. Asterisks, non-specific bands. GC B cell samples are same as in Fig. 2e. b Flag (MEF2B) IP followed by IB in SUDHL10 cells expressing Flag-HA-MEF2B-WT-isoA or missense mutants. Lamin B1, loading control. Quantifications were normalized to the immunoprecipitated MEF2B (HA). Fold changes relative to MEF2B-WT. Samples in the left panel are same as in Fig. 5a. c MEF2B IP followed by IB using nuclear extracts of isogenic OCI-Ly7 control (Neutral) and MEF2B missense mutant clones. IgG, IP negative control. Quantifications were normalized to the immunoprecipitated MEF2B. Fold changes were calculated relative to the neutral clones. Samples are same as in Fig. 5b. d Ubiquitination assay upon Flag (MEF2B) IP followed by IB of HEK293T cells co-transfected with expression vectors for Flag-HA-MEF2B-WT-isoA or missense mutants, 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. e MEF2B relative expression level, upon CHX treatment (12 h) in SUDHL10 cells expressing Flag-HA-MEF2B-WT-isoA (n = 13) or the missense mutants S324A (n = 6), S324E (n = 3), P325L (n = 8), EL (n = 3), R322C (n = 3) and P330L (n = 3). Numbers (n) refer to independent experiments. Complete CHX pulse-chase experiment in Supplementary Fig. 7b. Average fold change ± SD relative to MEF2B-WT-isoA (two-tailed unpaired T test). f MEF2B relative expression level, upon CHX treatment (12 h) in isogenic OCI-Ly7 control (Neutral; n = 6) and MEF2B-P325L mutant (n = 6) clones. Complete CHX pulse-chase experiment in Supplementary Fig. 7c. Average fold change ± SD relative to neutral clones of two independent experiments (3 neutral compared to 3 P325L clones in each experiment; two-tailed unpaired T test). The experiment in (a), (b), and (d) were repeated three times with similar results. Source data are provided as a Source Data file.

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