Pv3850

In vitro CSNK1A1 kinase assays were performed using assay conditions adapted from manufacturer recommendations (Recombinant CSNK1A1, PV3850, Thermo Fisher). All reactions were performed in a 50 μL volume for 90 minutes at 30°C. Assay buffer was composed of 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 2.5 mM DTT, 0.01% Triton X-100, 0.5 mg/mL BSA, 0.5 mM EGTA, 0.5 mM Na₃VO₄, 5 mM beta-glycerophosphate, 1 mM peptide (Supplementary Table 8), 170 ng of recombinant CSNK1A1, and 200 μM [γ-³²P]ATP (SA = 100-500 cpm/pmol). Reactions were terminated using 75 mM H₃PO₄ and spotted onto P81 phosphocellulose squares. Samples were washed four times in 75 mM H₃PO₄ for 5 minutes per wash and immersed in acetone for 5 minutes before drying. ³²P incorporation was assessed by Cerenkov counting.

AGO2^−/− cells were infected with MSCV retroviral constructs to stably express FH-AGO2^WT or FH-AGO2^5XA. FH-AGO2-expressing cells were seeded using 1.5×10⁷ cells per dish in 15 cm dishes with three dishes per cell line. Lysates were generated using methods similar to the co-immunoprecipitation assays, with the exception that 2 mL of lysis buffer was used per dish. Lysates were diluted with one volume of lysis buffer. FH-AGO2 was immunoprecipitated using 9 μg of anti-FLAG antibody (F1804, Sigma) and 150 μL of washed Dynabeads. Samples were rotated at 4°C overnight. Beads were washed three times with lysis buffer and then treated with lambda protein phosphatase (NEB) for 45 minutes. Beads were washed three times with lysis buffer and then resuspended in 100 μL reaction buffer composed of 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 2.5 mM DTT, 0.01% Triton X-100, 0.5 mg/mL BSA, 0.5 mM EGTA, 0.5 mM Na₃VO₄, 5 mM beta-glycerophosphate, 170 ng of recombinant CSNK1A1 (PV3850, Thermo Fisher), and 200 μM [γ-³²P]ATP (SA = 100-500 cpm/pmol). Reactions were incubated at 37°C for 2 hrs. Beads were separated and mixed with 50 μL of 2X Laemmli sample buffer. SDS-PAGE was performed, and gels were stained using SimplyBlue SafeStain (Invitrogen). ³²P signal was detected using a phosphor screen (GE Healthcare) and Typhoon FLA 7000 (GE Healthcare).