Supplementary Figure 1: The WD40 domain of Pan2 directly contacts the Pan2–Pan3 core complex.
From: The structure of the Pan2–Pan3 core complex reveals cross-talk between deadenylase and pseudokinase
(a) RNase assays with Pan2fl–Pan3fl-H10 using different 15mer homo-oligonucleotides, carried out as in Fig. 1c. Abbreviation: conc. (concentration). (b) Protein co-precipitation of H10Pan3CR (His-tagged Pan3 C-terminal region, residues 275–679) and Pan2fl (Pan2 full-length). The Nickel pull-down assay was performed on the cell lysate in the presence of 300 mM salt and samples were separated on 4-12% SDS-PAGE. (c) Left panel: protein co-precipitation of Pan3ΔN-H10 (His tagged Pan3 residues 226–679) and Pan2N-3C-C (fl Pan2 with a 3C protease cleavage site engineered after the N-terminal WD40 domain, between residues 333 and 334). The Nickel pull-down assay (His-Select resin, Sigma) was carried as described in panel a. Central panel: analysis of the Pan3ΔN-H10– Pan2N-3C-C sample after 3C protease cleavage (lane 1). The cleaved sample was purified on an ion exchange column (AEC), which separated the excess of 3C protease (lanes 2-4) from three polypeptides which co-eluted in the same peak: Pan2N (residues 1–333), Pan2C (residues 334–1115) and Pan3ΔN-H10 (lane 5). The three peptides also co-eluted by size exclusion chromatography (SEC,lane 9). Right panel: chromatogram of the size-exclusion chromatography. Highlighted in red is the peak fraction corresponding to lane 9 of the gel on the left. Abbreviations: 3C-prot. (3C-protease), A280nm (Absorption at 280 nm).
