Briefly, 5 µg of CaCo2 nuclear extract were incubated with the binding buffer consisting of 25 mM HEPES (pH 7.6), 5 mM MgCl2, 34 mM KCl, 2 mM DTT, 2 mM Pefablock (Roche Diagnostics GmbH, Mannheim, Germany), 0.5 µL aprotinin (2.2 mg · mL−1, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), 50 ng poly (dl-dC) and 80 ng bovine serum albumin (PAA Laboratories GmbH, Cölbe, Germany).
A binding buffer consisting of 20 mM sodium phosphate, 0.15 M sodium chloride, pH 7.4 was used.
The bead-immobilized His6-PIAS3 was incubated for 4 h at 4°C with GST-NR2E3 in 1 ml of binding buffer consisting of 50 mM Tris HCl (pH 7.6), 150 mM NaCl and 10 mM imidazole.
For the DNA-binding experiments 10 ng (3 nM) of biotinylated atoD probe were used in binding buffer consisting of 10 mM Tris-HCl pH 7.4, 50 mM NaCl, 1 mM EDTA, 4 mM DTT and 5% (v/v) glycerol, as previously described [ 5].
Nuclear extract (20 μg) was incubated at 22 °C for 30 min with NF- κB P-labelled oligonucleotide and gel shift binding buffer consisting of 2.5 mmol l 1 DTT, 0.25% Tween-20, and 0.25 mg ml 1 poly dI):poly dC).
After a 15 minute incubation step with the permeabilization buffer cells were centrifuged and resuspended in 100 μl of this buffer adding 30 μl of RNase A (1mg/ml, R4875, Sigma-Aldrich, Hamburg, Germany) and 1 ml of binding buffer consisting of HBSS (Sigma-Aldrich, Hamburg, Germany), 10% FCS (PAN™ Biotech GmbH, Aidenbach, Germany) and 0.01 M HEPES (Biochrom, Berlin, Germany).
The binding buffer consisted of 1-0.1 MnCl2Cl2, 5 mM MnCl2, 80 mM glutamate potassium glutamate, 10 mM mercaptoethanol, 10% DMSO, and 35 mM MOPS (pH 7.2).
The phosphate binding buffer consisted of 20 mM phosphate (pH 7.4), 10% glycerol, 3 mM MgCl2, 17 mM KCl, 2.5 μM DTT, and 50 MM–2 M NaCl.
The binding buffer consisted of 20 m m Tris HCl pH 7.5 20°CC), 5 m m MgCl2, 100 m m KCl, 10 µ m ZnCl2, 0.5 m m Tris 2-carboxyethyl phosphinee hydrochloride, 100 µg/ml BSA and 1 µg/ml polydIdC.
For βARs, the membrane pellets were resuspended and resedimented in a buffer consisting of 125 mM sucrose, 6 mM MgCl2, 50 mM Tris-HCl (pH 7.5), whereas for m2AChR binding, we maintained the same sodium-phosphate buffer used for the original homogenization.
The cells were collected by centrifugation and re-suspended into 20 ml of binding/washing buffer consisting of 20 mM Tris HCl (pH 8.0), 500 mM NaCl, and 20 mM imidazole HCl (pH 8.0).
