Figure 2. The K700E Mutation Specifically Impairs Association with SUGP1.
(A) Total RNA was extracted from K562 parental cells and CRISPR/Cas9 engineered K562 cells expressing mono-allelic His6-FLAG-tagged WT or K700E SF3B1, followed by RT-PCR of the cryptic 3′ss (open arrowheads) and canonical 3′ss (solid arrowheads) of two select genes (GCC2 and KANSL3).
(B) SF3B1-associated proteins were purified from CRISPR/Cas9 engineered K562 cells expressing mono-allelic His6-FLAG-tagged WT or K700E SF3B1 using the large-scale affinity purification protocol, followed by SDS-PAGE and staining with QC Colloidal Coomassie Stain (Bio-Rad). M, Precision Plus Protein marker (Bio-Rad).
(C) Mass spectrometry results of three candidate proteins differentially associated with WT (W) and K700E (K) SF3B1, with the second and third columns indicating the numbers of unique peptides and the fourth column the peptide ratio.
(D and E) SF3B1-associated proteins were purified from CRISPR/Cas9 engineered K562 cells expressing mono-allelic His6-FLAG-tagged WT (W) or K700E (K) SF3B1 using the small-scale protocol, and then resolved by SDS-PAGE, followed by silver staining (D) or western blotting (E). M, Precision Plus Protein marker (Bio-Rad).
(F) Mass spectrometry results of SF3b subunits (as well as SUGP1) associated with WT and K700E SF3B1. Note that in the K700E SF3B1 sample there was one unique peptide that belongs to a novel isoform (fragment) of SF3B2, but this single peptide was not included in the total number of unique peptides of SF3B2, which does not affect our conclusion.