Two hours after stimulation with 100 nM PMA (Sigma-Aldrich, St. Louis, MO, USA), endogenous nuclear protein fraction was extracted from Jurkat cells using the Cell Lytic NuCLEAR Extraction Kit (Sigma-Aldrich, St. Louis, MO, USA). Nuclear extracts were stored at -80°C until use. 5 μg were used per lane. EMSAs were performed utilizing Odyssey EMSA Buffer Kit (Li-Cor Biosciences, Lincoln, NE, USA) in a composition of 10 mM tris, 50 mM potassium chloride, 1.75 mM dithiolthreitol (DTT), 0.25% tween, 5% glycerol, 0.1 mM EDTA, and 2 mg/ml bovine serum albumin. Buffer ingredients were incubated for 35 minutes at room temperature prior to addition of oligonucleotides, followed by 30 minutes of incubation under light-protected conditions after the addition of oligonucleotides (Eurofins Genomics, Ebersberg, Germany). EMSAs were carried out using double stranded oligonucleotide sequences given as sense strand sequence: GRK6 promoter sequence containing a putative CREB binding site CREB GRK6: 5’-AGGAAGAGGTGACGTGATTGGTCCCGC-3’ (position -365/-338 in relation to translation starting point ATG), CREB positive control, derived from CREB1 consensus binding site: 5’-AGAGATTGCCTGACGTCAGAGAGCTAG-3’, and an oligonucleotide containing a mutated CREB1 binding site as CREB negative control: 5’-AGAGATTGCCTGTGGTCAGAGAGCTAG-3’. Sequences suspected as putative transcription factor binding sites are underlined, mutated bases are underlined twice. Oligonucleotides were labelled with dye DY682 at their 5’ end. Competition was carried out using similar unlabelled oligonucleotides 200 times in excess. Super shift was conducted using 2 μl rabbit anti-CREB1 p43 polyclonal antibody (antibody registry ID AB_91283, catalogue no. AB3006 Chemicon, Tenecula, CA, USA). Pre-incubation with antibody was carried out for 35 minutes at room temperature prior to the addition of oligonucleotides. Electrophoresis was performed with 5% native polyacrylamide gels. Odyssey Classic Imaging System (Li-Cor Biosciences, Lincoln, NE, USA) was used for detection. Images were cropped using the GNU Image Manipulation Program (Free Software Foundation, Boston, MA, USA).

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