Expression and functional characterization of SF3B1 in-frame deletions. (A) Fold change of mxSF3B1 variant transcript relative to WT (calculated as 2^−ΔΔCT) in HEK293FT cells transfected with mxSF3B1 WT, K700E, K700del, Q698del, and K700N complementary DNA constructs. Data are represented as mean ± standard error of the mean, n = 3. (B) Incorporation of SF3B1 variants in SF3b complex is shown through western blot analysis of 5 different complex partners after coimmunoprecipitation using α-HA beads. Input cell lysates are shown in the left panel, and the eluted IP samples are shown in the right panel. Schematic of SF3b in U2 complex is also shown with pre-mRNA branch site labeled as “A.” (C-D) Schematic of ZDHHC16 minigene labeled with ss’s is shown with cryptic AG (red) and canonical AG (green) underlined. Total RNA isolated from HEK293FT cotransfection of mxSF3B1 variants (WT, K700E, K700del, Q698del, K700N) with any of the 3 different ZDHHC16 minigenes (E9-I9-E10, −3T>G, −24C>G) (C) or with any of the 2 different ZDHHC16 minigenes (−30A>G, −33 to −35 AAA>GGG) (D) was used for reverse transcription PCR and visualized by ethidium-bromide stained 2.5% agarose gel. Sequences of different minigenes with specific mutations highlighted in bold are shown for respective gels. Spliced controls of canonical (CJ) and aberrant junction (AJ) are in lanes 1 and 2, marker is denoted as M, and nontransfected control is shown as NT. Spliced product for junction in exon 10 is shown as E10J. (E) Effects of splicing inhibitor E7107 treatment on viability of primary CLL patient cells (K700del, Q698del, SF3B1-K700E, SF3B1-WT 1, and SF3B1-WT 2) measured through MTS assay. E7107 concentration is plotted in log scale in the x-axis; % absorbance in the colorimetric assay is represented as % growth in the y-axis. Data are represented as mean ± standard deviation, n = 3.