Fig. 5: Phosphorylation-deficient MEF2B mutants display enhanced binding affinity to SWI/SNF complex and increased transcriptional activity.

a Detection of MEF2B/SMARCA4 interaction by Flag (MEF2B) immunoprecipitation (IP) followed by immunoblotting (IB) from SUDHL10 cells expressing Flag-HA-MEF2B-WT-isoA or missense mutants (EL, S324E + P325L). Lamin B1, loading control. Quantifications were normalized to the immunoprecipitated MEF2B (HA). Fold changes relative to MEF2B-WT. b MEF2B IP followed by IB from isogenic OCI-Ly7 control (Neutral) and MEF2B missense mutant clones. IgG, IP negative control. Quantifications were normalized to the immunoprecipitated MEF2B. Fold changes relative to the neutral clones. c Luciferase activity driven by synthetic wild-type (3xMEF2-WT) or mutated (3xMEF2-Mut) MEF2 binding sites, as measured in HEK293T cells transfected with empty vector (EV) or plasmids expressing MEF2B-WT-isoA (n = 15, 5 experiments) or the missense mutants S324A (n = 15, 5 experiments), S324E (n = 6, 2 experiments), P325L (n = 15, 5 experiments), EL (n = 6, 2 experiments), R322C (n = 9, 3 experiments) and P330L (n = 9, 3 experiments). Data are shown as average fold change ± SD relative to MEF2B-WT-isoA of indicated independent experiments, each performed in triplicates. Significance assessed by two-tailed Mann-Whitney test. d Flag (MEF2B) IP followed by IB from SUDHL10 cells expressing Flag-HA-MEF2B-WT-isoA, MEF2B-WT-isoB or isoform-switch mutants. Lamin B1, loading control. Quantifications were normalized to the immunoprecipitated MEF2B (HA). Fold changes relative to MEF2B-WT-isoA. e MEF2B IP followed by IB from isogenic OCI-Ly7 control (Neutral) and MEF2B isoform-switch mutant clones. IgG, IP negative control. Quantifications were normalized to the immunoprecipitated MEF2B. Fold changes relative to the neutral clones. f Luciferase activity of synthetic WT or mutated 3xMEF2-luc reporter in HEK293T cells transfected with EV or plasmids expressing MEF2B-WT-isoA (n = 18, 6 experiments), -isoB (n = 18, 6 experiments) or isoform-switch mutants T240fs (n = 9, 3 experiments), P243fs (n = 9, 3 experiments), P256fs (n = 9, 3 experiments), L260fs (n = 9, 3 experiments), L269fs (n = 9, 3 experiments), P273fs (n = 6, 2 experiments), T274fs (n = 9, 3 experiments) and P287fs (n = 9, 3 experiments). Data shown as average ± SD relative to MEF2B-WT-isoA of indicated independent experiments, each performed in triplicates (two-tailed Mann-Whitney test). The experiments reported in (a) and (d) were repeated three times with similar results. Source data are provided as a Source Data file.