Cells then were rinsed with ice-cold PBS twice and collected into

Cells then were rinsed with ice-cold PBS twice and collected into 100 mM Tris-HCl (pH 9.4), 10 mM DTT and incubated for 15 min at 30��C and centrifuged for 5 min at 2000 g. Cells were washed sequentially with 1 Bicalutamide ml of ice-cold PBS, buffer I (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5), and buffer II (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5). Cells were then resuspended in 0.3 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1�� protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and sonicated three times for 10 s each at the maximum setting (Branson, Digital Sonifier) followed by centrifugation for 10 min.

Supernatants were collected and diluted in buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8) followed by immunoclearing with 2 ��g sheared salmon sperm DNA, 20��l pre-immune serum and protein A-sepharose (45 ��l of 50% slurry in 10 mM Tris-HCl, pH 8, 1 mM EDTA) for 2 h at 4��C. Immunoprecipitation was performed overnight at 4��C with anti-HIF1�� antibody (Novus Biologicals, CO, USA), or with control IgG antibody (Pierce). After immunoprecipitation, 45 ��l protein A-Sepharose and 2 ��g of salmon sperm DNA were added and the incubation continued for 1 h. Precipitates were washed sequentially for 10 min each in TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8, 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8, 500 mM NaCl), and buffer III (250 mMLiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8).

Precipitates were then washed three times with TE buffer and extracted three times with 1% SDS, 100 mM NaHCO3. Eluates were pooled and heated at 65��C for at least 6 h to reverse the formaldehyde cross-linking. DNA fragments were purified with a Montage PCR Kit (Millipore, Germany). PCR was performed using PCR Master (Roche Diagnostics Cilengitide GmbH, Mannheim, Germany) with the following primers: 5��-AGATCTTTCGTTAAACCCCTGGTCCG-3�� and 5��-CTCGAGTCATGTCCTTTCTCACGTCCA-3��, detecting the region ?33 to?493 in TSP-1 promoter (35 cycles). The PCR products were separated by electrophoresis in 2% agarose gel. Electrophoretic Mobility Shift Assay Nuclear extracts from miHIF1�� or mock-transfected and non-transfected U937 cells were obtained as described above. Synthetic oligonucleotides (biotin-labelled) were synthesized (TIB MOLBIOL GmbH Berlin, Germany) and used as probes in electrophoretic mobility shift assays (EMSAs). Analysis revealed what appeared to be a hypoxic response element (HRE) motif (-62 TCA CAA TCT GGA CGT GAG AAA GGA CAT-35) in the TSP-1 promoter. A mutated probe-binding site (TSP-1 mt: TCA CAA TCT GGA AAA AAG AAA GGA CAT) or excess unlabelled probes (XS) were used as controls.

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