Superose 6 3.2 300 column

For the size-exclusion chromatography (SEC) assays in Figure 2B, purified Pab1 (36 μM final concentration in the case of 90A, 24 μM in the case of 70A and either 36 μM (“3x 30A/Pab1”), 24 μM (“2x 30A/Pab1”) or 12 μM (“Pab1” and “Pab1/30A”) final concentration in the case of 30A was mixed with the respective RNA (90A oligo RNA at 12 μM final concentration, 70A oligo RNA at 12 μM, 30A oligo RNA at either 36 μM [“3x 30A/Pab1”], 24 μM [“2x 30A/Pab1”] or 12 μM [“Pab1/30A”]) final concentration and incubated at 4°C for 30 min (all oligo(A) RNAs were procured from Ella Biotech). Pan2cat-Pan3 (12 μM final concentration) was added to these reactions and again incubated for 15 min at 4°C in a total injection volume of 30 μl in SEC buffer (20 mM HEPES/NaOH pH 7.5, 50 mM NaCl, 100 mM potassium acetate, 5 mM magnesium acetate, 2 mM DTT). Complex formation was assayed by comparing the retention volumes in SEC on a Superose 6 3.2/300 column (GE Healthcare). Composition of the SEC peak fractions were analyzed by SDS-PAGE on 4%–12% NuPAGE gradient gels (Thermo Fisher Scientific) and visualized by Coomassie staining. For better comparison, each individual chromatogram was normalized by its maximal absorption value and plotted in R using the tidyverse collection of R packages.

N-terminal myc-tagged p63 isoforms and mutants were expressed in vitro using rabbit reticulo lysate expression system (Promega) as described previously^{8 (link)}. Lysates were centrifuged at 16,100 × g for 15 min at 4 °C. Analytical SEC was performed at the Äkta purifier system in phosphate buffer (50 mM sodium phosphate pH 7.8, 100 mM NaCl) at 4 °C using a Superose 6 3.2/300 column (GE Healthcare) (injection volume 50 μL; flow rate 50 μL/min; fraction size 50 μL). The SEC fractions were collected and analyzed via western blotting.