SR-B1 is a lipoprotein receptor, and we observed a significant decrease in the binding of BCG-lux to R6F cells expressing SR-B1 when binding was performed in the presence of serum (Fig. 2A, left panel).
Far Western blot analysis was carried out using 250 ng of His-tagged NPM/B23 as bait and ligand binding was performed in 5% milk powder in TBST (40 mM Tris-HCl pH7.5, 300 mM NaCl, 0.2% Tween20), with 1 µg/ml GST-Plk2-PBD, GST-Plk2-PBD mutant or GST alone for 6 h at 4°C.
DNA-protein binding was performed in 30 ul reaction mixture containing 100 mM KCl, 1 mM MgCl2, 10 µM ZnSO4, 10 mM Tris, pH 7.5, 4% glycerol, 0.1% Triton X-100, 1 mg/mL BSA, 1 µg of poly dIdC /poly dAdT), 0.5 mM DTT and protease inhibitor cocktail mini tabs (Sigma).
In vitro binding was performed in 500 µl of buffer A (50 mM Hepes-NaOH (pH 7.5), 3 mM MgCl2, 2 mM CaCl2, 3 mM EGTA, and 80 mM KCl) containing 1% Triton X-100 at 4°C for 3 h.
Protein binding was performed in 20-µL reactions containing 32 pmol of SR protein or BSA, 80 fmol of uncapped IgM M1-M2 RNA labeled with all four NTPs, in reaction buffer conditions with final concentrations of 1.6 mM MgCl2, 20 mM HEPES-KOH pH 7.3, 0.5 mM ATP, 20 mM creatine phosphate, and 60 mM KCl.
Where indicated, binding was performed in the presence of additives such as 10% serum, 1∶100 diluted rabbit anti-mouse SR-B1 polyclonal antibody (NB 400-134, Novus Biologicals), 100µg/ml LDL, 50% bronchoalveolar lavage fluid (BAL), or methyl-β-cyclodextrin (MβCD, Sigma), respectively, at the indicated concentrations.
The key advantage of our system is that screening for pair binding is performed in parallel for all aptamers on the array simultaneously.
Furthermore, when the SP-stimulated (1 µmol/L) [35S]GTPγS-binding was performed in the presence of ezlopitant (0.1 nmol/L–100 µmol/L) the binding was potently inhibited in both groups (water group, IC50 = 1.5±0.5 nM, Figure 8C: sucrose group, IC50 = 61±3.1 nM, Figure 8D).
All individual bindings were performed in duplicate with at least 12 data points per curve.
Aven binding was performed as in panel B. PRMT1 depletion was confirmed by immunoblotting.
Subsequently displacement binding was performed for each ligand in the presence and absence of a constant concentration of T30 (100 nM).
