Extended Data Fig. 1: Constructs and complex production.
From: Cryo-EM structures of PP2A:B55–FAM122A and PP2A:B55–ARPP19
a. Construct schematic. ARPP19 constructs phosphorylated as indicated. Most highly conserved FAM122A residues are shown. b. Schematic describing the production of PP2A:B55 and PP2A:B55-inhibitors for structural and biophysical studies. c. Immunoblots of purified PP2A:B55 (above, SDS-PAGE), with and without NaOH treatment, using antibodies detecting methylated PP2Ac (BioLegend, Cat# 828801), PP2Ac (Millipore, Cat# MABE1783) and B55 (Cell Signaling Technologies, Cat# 2290 S). d. Size exclusion chromatography (SEC) chromatogram of PP2A:B55; peak 1 corresponds to PP2A:B55; peak 2 is excess free B55. e. PP2A:B55 activity assay using DiFMUP as a substrate; PP2A:B55 concentrations 0.3125-20 nM; 2.5 nM concentration highlighted in red. f. Same as e (2.5 nM concentration) with and without the PPP inhibitor microcystin-LR (3.75 nM concentration). g. SDS-PAGE of the PP2A:B55-tpARPP19 used for Cryo-EM grid preparation and data collection. h. SDS-PAGE of the PP2A:B55-FAM122A used for Cryo-EM grid preparation and data collection. Results (c-d and g-h) representative of 3 independent experiments.
