Fig. 3.

In-cellulo evaluation of the aberrant splice pattern of SF3B1 mutants. A. Protein expression analysis of exogenous flagged-SF3B1 and endogenous SF3B1 in transfected HEK293T cells by immunoblotting with anti-Flag and anti-SF3B1, respectively. ß-actin antibody was used as a loading control. B. Effect of the various mutations of SF3B1 on the AG’/AG transcript expression ratio of DPH5, DLST, ENOSF1 and ARMC9 in HEK293T cells. Ratios of expression levels of the alternative AG’ and canonical AG forms (AG’/ AG) were determined by RT-qPCR. The results are average of three replicates and are represented as mean ± sd. Paired t-test was used to generate the p-values comparing each condition to the SF3B1 wild-type vector transfection (SF3B1-WT): *p < 0.05; **p < 0.005; ***p < 0.0005. C. Heatmap of ratios of aberrant to canonical 3′ss junction expression (AG’/AG index) for a panel of previously validated SF3B1^Mut-aberrant splice events in HEK293T cells transfected with the SF3B1 wild-type or hotspot mutants (left panel), and examples of IGV visualizations of SF3B1^Mut-aberrant splice events (right panel).