Lack of significant recruitment of the GR P-S203 isoform did not appear to differ between these two genes (Fig. GR P-S226 recruitment differed among genes. Our findings indicate that GR phospho-isoforms selectively occupy GR target genes, and suggests gene specific requirements for GR phosphorylation in receptor-dependent transcriptional activation. and were performed on 0.05 ng total RNA, RT-PCR products were separated on a 3% agarose gel, visualized by ethidium bromide staining and photographed under UV light. Scanned images were quantitated using NIH Image and normalized to mRNA levels. 2.4 Chromatin Immunoprecipitation Cells were cultured in 15 cm dishes (25 ml of media) and treated with 100 nM Dex for the times indicated. A 2.5 ml aliquot of 10X Norisoboldine ChIP fixation buffer (50 mM Hepes pH 8.0, 1 mM EDTA, 100 mM NaCl, 11% formaldehyde) was added and incubated for 15 min at 4C. Formaldehyde was quenched by adding 2.5 ml Norisoboldine of 1 1.4 M glycine per plate and incubated for 1 min at 4C. Formaldehyde-containing media was removed and the cells rinsed once with ice cold PBS. PBS Rabbit polyclonal to HYAL2 (2 ml) was added to each plate. Cells were scraped into the PBS, placed into a 15 ml conical tube, and pelleted at 300xg for 5 min at 4C. Cell pellets were flash frozen on dry ice and stored at ?80 C. Pellets were resuspended in RIPA buffer (10 mM Tris pH 8.0, 1 mM EDTA, 140 mM NaCl, 5% glycerol, 0.1% sodium deoxycholate, 0.1% SDS, 1.0% Triton X-100, 1 mM PMSF) to a final volume of 2 ml. Cells were sonicated at 30 watts input power (approximately 55% amplitude) in one second bursts, followed by three seconds of cooling on ice, for a total of 4 min using a Branson 250 digital sonifier fitted with a 1/2 step disruptor horn and an 1/8 titanium tapered microtip. Lysates were transferred to microcentrifuge tubes, centrifuged at 15,000 rpm for 10 min at 4C. Supernatants were transferred to a 15 ml conical tube and diluted to 3.5 ml with RIPA buffer. A portion (5 l) was removed and kept as the input for the ChIP assay. Immunoprecipitation were performed by adding 10 l of total rabbit sera containing antibodies that recognized either, total GR, GR P-203, GR P-211 or GR P-226 along with 30 l of 50% protein A-sepharose CL4B beads (Sigma) equilibrated in RIPA buffer with 100 g/ml boiled sheared salmon sperm DNA to the soluble chromatin. Immunoprecipitations were carried out overnight at 4C. Beads were pelleted and washed 5-times with 1 ml of ice-cold RIPA buffer for 5 min at 4C. SDS/proteinase K (10 mM Tris pH 8.0, 1 mM EDTA, 0.5% SDS, 200 g/ml proteinase K) (110 l) was added to the tubes and placed at 55C for 4 h and then overnight at 65C to reverse the crosslinks. The remaining chromatin was purified using the Qiagen PCR purification kit and eluted in 45 l of dH2O. PCR was performed for 35 cycles on 4 l of DNA using standard Promega Taq DNA polymerase conditions. Products were separated on a 3% agarose gel, and visualized by ethidium bromide staining. The images were scanned and quantitated using NIH Image, and normalized to input. Sequences of each primer pair are: TAT F 5-CCCATAAATAACAGGAAGCCCAAG -3; TAT R 5-ATACCACCACACCCA GAAACCGAC -3; SULT F 5-CGTGTGAATGCTCTGTCCCATC -3; SULT R 5-GCTGAAGATTTTGTGTCCCAAGTG -3; GILZ F 5-GATACCA GTTAAGCTCCTGA-3; GILZ R 5-AGGTGGGAGACAATAATGAT-3; UPS F 5-TCCCAAACAGATAGCTTTCT-3 UPS F 5-AGCAGCAGCCCTACTATT A-3 Norisoboldine 3. Results 3.1 GR phospho-isoform expression in rat hepatoma cells As a prerequisite for examining phospho-GR recruitment Norisoboldine to endogenous target genes, we determined the kinetics of receptor phosphorylation over 12 hours in a rat hepatoma line expressing endogenous GR upon dexamethasone (Dex) treatment using antibodies that specifically recognize phosphorylation of GR at S203, S211 and S226 [9, 14]. GR phosphorylation at all three sites was observed (Fig. 1). The GR P-S203 and GR P-S226 antibodies recognized GR from both untreated and hormone-treated cells and both sites show.