Because the driving force ([A]1 ? [A]2) remained virtually unchanged in the course of the described experiments, the relative changes in F correspond to similar changes in P. Cell culture. Pulmonary microvascular endothelial cells (PMEC) were isolated Ganetespib OSA as described by Lips et al. (26) with Dynabeads coated with Bandeiraea simplicifolia lectin. HL-1 cells were kindly provided by Dr. W. C. Claycomb (Louisiana State University Health Science Center, New Orleans, LA). NIH3T3 fibroblasts were from the German Resource Center for Biological Material (DSMZ, Braunschweig, Germany). NS20Y cells were from PAA (Pasching, Austria) and HMVEC-L from Lonza (Basel, Swizerland). For hypoxia experiments, cells were exposed for 6 h to either normoxia (21% O2, 5% CO2, 74% N2) or hypoxia (1% O2, 5% CO2, 94% N2).

RNA isolation and real-time RT-PCR. Total RNA was isolated with the RNeasy mini kit (Qiagen, Hilden, Germany), and genomic DNA was removed by treatment with DNase (Invitrogen, Karlsruhe, Germany). Superscript RNase H? reverse transcriptase was used for reverse transcription. Real-time quantitative PCR was performed as described by Pfeil et al. (31). Primer sequences are specified in Table 1. Sequencing of the PCR products was done by MWG Biotech (Ebersberg, Germany). Table 1. Primers used for RT-PCR Analysis of intermedin gene promoter reporter activity. IMD gene sequences from human and mouse were retrieved from GenBank and analyzed with the TRANSFAC database and Perl-based scripts. A luciferase reporter construct, pGL2�C2.7 kb IMD promoter-luc (pGL2-Enhancer luciferase vector, Promega, Mannheim, Germany), which contains a 2.

7 kb (?1 ~ ?2650) proximate region of mouse IMD gene promoter, was cotransfected with a HIF-1�� expression construct (GeneCopoeia, Germantown, MD) in HEK293T cells. Cells were transfected with 4 ��g of pGL2�C2.7 kb IMD promoter-luc plasmid, together with 2, 4, 8, or 16 ��g of the HIF-1�� expression vector plasmid, by the calcium phosphate precipitation method. Five-hundred nanograms of a pCMV-��-galactosidase vector were cotransfected to monitor transfection efficiency. The reporter activity is expressed as the ratio of relative luciferase unit to ��-galactosidase activity. RESULTS IMD is expressed by pulmonary endothelial cells. The library screen identified nine antibody clones that recognized IMD, but not AM, in ELISA.

Two of these (clones “type”:”entrez-protein”,”attrs”:”text”:”AbD06980.1″,”term_id”:”86572423″,”term_text”:”ABD06980.1″AbD06980.1 and “type”:”entrez-protein”,”attrs”:”text”:”AbD06988.1″,”term_id”:”86572431″,”term_text”:”ABD06988.1″AbD06988.1) were suitable for immunofluorescence GSK-3 labeling of formaldehyde-fixed tissue. Specificity of these anti-IMD antibodies was shown in dot blot and Western blot analysis.