Typhoon fla 7000 phosphoimager

Preparation of recombinant Drosophila CCR4/CAF1 heterodimer and deadenylation assay with the enzyme was previously described (Niinuma et al. 2016 (link)). Deadenylation reaction to estimate the inhibitory effect by AMP, ADP, and ATP contained 4.2 µL of water, 3 µL of 1.5 µM CCR4/CAF1 heterodimer, 3 µL of 5× lysis buffer, 1.5 µL 10 mM DTT, 0.3 µL of 40U/µL RNasin plus (Promega), 1.5 µL of ∼5 nM noC-A₄₀, and 1.5 µL of 10× AMP, ADP, or ATP. The mixture was incubated at 25°C, and 3 µL of it was collected at each time point. The equal volume of formamide loading dye was added to the collected sample immediately. Then, the sample was electrophoresed on 5% denaturing polyacrylamide gel and analyzed by PhosphoImager (Typhoon FLA 7000, GE Healthcare).

Brain sections on slides were removed from the −80 °C freezer and thawed at room temperature for 5 min before rehydration in PBS (pH 7.4) for another 5 min. Brain sections were then incubated with a range of concentrations of tritiated ligands ([³H]RAGER, [³H]FPS-ZM1, or [³H]azeliragon) and unlabeled ligands. Incubation was conducted at room temperature, and incubation times were determined by equilibrium experiments; 30 min incubations were used for [³H]RAGER and [³H]FPS-ZM1, while 60 min was used for [³H]azeliragon. All sections were washed for 3 × 2 min with PBS at 4 °C and then rinsed in dH₂O for 30 s at 4 °C to remove unbound radioactivity. Finally, slides were dried under the continuous airflow for 30 min before exposure to a high-resolution phosphoimaging plate for 2 weeks. The exposed plate was scanned using a GE Typhoon FLA 7000 phosphoimager. Image analysis was performed using ImageQuant (Molecular Dynamics) software. Regions-of-interest were drawn and converted to disintegrations per minute (DPM)/μg protein using the Amersham standards, and then using the individual ligand’s molar activity converted to nmol/μg protein.

‘Top’ and ‘bottom’ DNA oligonucleotides of 12, 22 and 32 bp length were purchased from IDT. The top strands were 5′-end labeled with using T4 Polynucleotide Kinase (New England Biolabs). The labeled strands were then annealed, in an excess concentration, with their corresponding complementary DNA strands and purified from a gel. The DNA constructs were incubated for 3 hours on ice (∼ C) with a gradient of concentrations of Egr1 in binding buffer. The samples were loaded on a native 12% polyacrylamide in 1× TBE gel while running, to ensure fast entry of the complex to the gel's wells. Gels were run for 45 min at 200 V, ensuring that the temperature of the running buffer is maintained below 10°C. Gels were dryed (BioRad, 583) and imaged using a GE Typhoon FLA7000 phosphoimager. The fraction of bound protein was estimated using ImageLab. Curves for the fraction bound vs. Egr1 concentration were fitted to a hyperbolic binding equation to extract the dissociation constant (Supplementary Figure S6F). To validate that [DNA] [Egr1], we conducted experiments at different DNA concentrations that resulted in the same binding curves. The results shown are an average of multiple gels, and the error bars are the standard error of the mean (Supplementary Figure S6G).