Extended Data Fig. 1: Biochemical characterization of recombinant SMG1-SMG8-SMG9.
From: InsP6 binding to PIKK kinases revealed by the cryo-EM structure of an SMG1–SMG8–SMG9 complex
Biochemical characterization of recombinant SMG1-SMG8-SMG9 complex (SMG1c). (a) Size-exclusion chromatography assay showing the formation of homogeneous SMG1-SMG8-SMG9 ternary complex. After expression in mammalian cells, the complex was purified by size-exclusion column (Superose 6 Increase 10/300 GL, void volume of 8.0 ml). Top panel: chromatography profile, with absorbance at 280 nm and 260 nm shown as blue and purple traces, respectively. Bottom panel: Coomassie-stained SDS-PAGE gel, with samples from each fraction that eluted between 8.5–16 ml; the SMG1c eluted between 13–15 ml. (b) Mass spectrometry (MS) analysis of purified SMG1. Graphical representation shows the result of an in-gel peptide mass fingerprinting experiment. Identified peptides are indicated as red dashes at the corresponding position in the SMG1 sequence (horizontal axis) and are plotted against their intensity, as detected by MS (vertical axis). The uniform distribution of detected peptides over the entire amino acid sequence indicates that the entire SMG1 polypeptide is present in the purified sample.
