Fig. 3: Variable impairment of SETBP1 degradation via proteasome and autophagy pathways is associated with partial reduction of ubiquitination.

a Fibroblasts derived from healthy controls and patients carrying SETBP1 variants outside the degron were treated with a translation inhibitor, cycloheximide (CHX; 100 μg/ml) or vehicle control DMSO for 4 h. Immunoblots of whole cell lysates probed with an anti-SETBP1 antibody were shown. β-actin was used as a loading control. Results are representative of three independent experiments. b Relative fluorescence intensity of YFP-tagged SETBP1 variants overexpressed in HEK293T/17 cells treated with translation inhibitor cycloheximide (CHX; 50 µg/ml; top), proteasomal degradation inhibitor MG132 (5 µg/ml; middle), or autophagy inhibitor Bafilomycin A1 (BafA1; 100 nM; bottom). An equal volume of DMSO was used as a vehicle control. Fluorescence intensity was measured for 24 h with 3-h intervals and normalised to an mCherry transfection control. Values are expressed relative to t = 0 h and represent the mean ± SEM of three independent experiments, each performed in triplicate (^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001; two-way ANOVA and a post-hoc Dunnett’s test). Details of statistical tests and p-values are provided in Source Data 4. c Immunoprecipitation of FLAG-tagged SETBP1 wild type and variants using FLAG-conjugated magnetic agarose and blotted with an anti-FLAG, anti-SETBP1 or anti-ubiquitin antibody. β-actin was used as a loading control. d Quantification of SETBP1 (top), ubiquitin (middle) in FLAG-IP fractions for inherited (outside degron, grey) and SGS variants (orange, left), and de novo variants outside the degron (right). Ratio of ubiquitin/SETBP1 in the FLAG-IP fractions was plotted (bottom). Bars represent the mean ± SEM of three independent experiments (^*p < 0.05, vs WT; one-way ANOVA and a post-hoc Dunnett’s test). Details of statistical tests and p-values are provided in Source Data 4. Source data are provided as a Source Data file.