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). Electrophoretic mobility shift assay (EMSA) Nuclear extracts for EMSA were prepared with nuclear and cytoplasmic extraction reagents (NE-PER; Pierce). The supernatant fraction containing the cytoplasm and the nuclear extract were prepared separately

nuclear protein and electrophoretic mobility shift assay (EMSA) Cells were plated at a density of 6×10 5 cells/cm 3 in a 10 cm dish. The culture medium was removed when the cells reached 90% confluence and 1 ml PBS was added to cover the cells

mobility shift assay (EMSA) Nuclear protein extracts from ovaries or corpora lutea were prepared by extracting nuclei with the following buffer: 0.42 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 1 mM dithiothreitol, 25% (v/v) glycerol, 0.5 mM phenylmethylsulfonyl

), electrophoretic mobility shift assay (EMSA), and immunocytochemistry (ICC) Transfected or infected cells in a time-dependent manner were processed for WB, EMSA, and ICC as described previously ( Muyan et al . 2001 , Yi et al . 2002 a ). For WB, proteins were

performed an electrophoretic mobility shift assay (EMSA) experiment using kJHRE (GGCCTCCACGTG) in the Krh1 promoter of Tribolium as a probe. In the RFP-expressing cells, the nuclear extracts from normal cells, or DMSO- or 20E-treated cells could not

. Differences were considered significant if P <0.05. Nuclear extracts and electrophoretic mobility shift assay (EMSA) The HEK293 cells were seeded in 10 cm tissue culture dishes (Nunc) in RPMI 1640 medium supplemented with

870 Da and 70 950 Da for the AR-DBD and AR-NTD-DBD polypeptides respectively. Protein–DNA interactions – electrophoretic mobility shift assays (EMSA) Oligonucleotides (Table 1 ) were end-labelled with 33 P γ

. Electrophoretic mobility shift assay (EMSA) for the SR region was also prepared using nuclear extracts with non-transfected CV1 cells. A 25- to 50-fold molar excess of cold oligonucleotides for 2G, 4G (sense 5′-cagtatgaattttcaatggggagatgcttttcagataagaaa-3′ and

and detected using the streptavidine alkaline phosphatase substrate. Nuclear extracts (2.5 μg) from transfected TSA-201 cells were preincubated at room temperature in a 20 μl reaction using LightShift Chemiluminescent EMSA kit (Pierce, Rockford, IL

before being shocked. At T0, 50% horse serum was added to the cells for 2 h. The cells were then grown in DMEM without serum for 2 days and harvested at various circadian times. Electrophoretic mobility shift assays (EMSAs