SF3B1 interacts with phosphorylated Pol II that regulates RBM39 poison exon inclusion. (A) GO analysis of the identified proteins from Pol II immunoprecipitation (IP)-mass spectrometry. Pathways with by -log (FDR) and enrichment >5 are shown. (B) Protein-protein interaction network of 66 RNA splicing proteins identified in the Pol II IP-mass spectrometry. (C-E) PCR analysis for RBM39 alternative splicing upon SRSF1 (with shSRSF1.1 and shSRSF1.2) (C), SRSF2 (with shSRSF2.1 and shSRSF2.2) (D), and of SF3B1 (with shSF3B1.1 and shSF3B1.2) (E) silencing in NALM6 cells. PCR reactions were performed for detection of RBM39 splicing event in indicated cells with EHT1610 (2 μM) treatment for 4 hours. (F) Western blot analysis following IP for Pol II in NALM6 cells. (G-H) The NALM6 cells were treated with DMSO, EHT1610 (5 μM), and THAL-SNS-032 (500 nM) for 4 hours, followed by SF3B1 IP-mass spectrometry. Scatter plot showing the differential proteins in comparison with DMSO and EHT1610 (G) or DMSO and THAL-SNS-032 (H). (I-J) Western blot analysis following IP for SF3B1 in NALM6 cells treated with EHT1610 (I) or THAL-SNS-032 (500 nM) (J) for 4 hours. (K-L) Immunofluorescence analysis of p-Ser5 Pol II and SF3B1 in NALM6 cells treated with DMSO or EHT1610 (5 μM) (K). Scale bars depict 5 microns. Quantification of Manders' colocalization coefficient values between SF3B1 and p-Ser5 Pol II Immunofluorescence signal (L) (See methods). ∗∗∗P < .001.