Fig. 3: SF3B1K700E-specific UBA1ms compromises protein stability.
A Maps for ORF cDNA in pcDNA3.1 + /C-(K)-DYK plasmids for UBA1 transcript variant 1 (WT) and UBA1ms (MS) and experimental design for evaluation of UBA1ms in HEK293T cells. B qPCR analysis of UBA1 transcript levels in HEK293T cells 48 h following transfection with WT or MS relative to pcDNA3.1 control transfected cells. The fraction of UBA1 splice forms is shown within bars (n = 3). Mean ± SEM relative fold change. Two-way ANOVA with Tukey’s multiple comparisons test. C Immunoblot analysis and quantification of D FLAG-tagged and E UBA1a/b protein levels in HEK293T cells 72 h after transfection with WT, MS or control. Actin was used as a loading control and the highest relative FLAG and lowest relative UBA1a/b signals were used to normalize results (n = 4). Mean ± SEM relative signal intensity. Welch ANOVA with Dunnett’s multiple comparisons test. F Representative immunoblot analysis and G quantification of FLAG-tagged protein levels in HEK293T cells 72 h post-transfection with WT, MS, or control plasmids and treatment with 50 µg/ml cycloheximide (CHX) for the indicated time points (n = 3). Actin was used as a loading control and signals were normalized to relative signals at 0 h for each group. Mean ± SEM relative signal intensity, interpolation of a one-phase decay non-linear regression curve (dotted line). H Representative immunoblot analysis and I quantification of FLAG-tagged UBA1ms and cMYC protein levels in HEK293T cells 72 h post-transfection with the MS plasmid and treatment with 50 µg/ml cycloheximide (CHX) and 10 µM MG-132 for 6 h (n = 4). Actin was used as a loading control and signals were normalized to untreated controls. Mean ± SEM relative signal intensity. Two-way ANOVA with Tukey’s multiple comparisons test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns not significant.
