The degradation of Hsp90 protein and HSP90 mRNA are not explicitly regulated by heat shock in the model

ld mounting medium. The samples were observed on an LSM 510 Meta confocal microscope, and data were processed using the software provided by the manufacturer or Image J software. HEPES, 0.2 mM EDTA, 25% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and complete protease inhibitors. 11885967” Protein concentrations were determined by Bio-Rad protein assay kit. The EMSA probes were 21278085” double-stranded oligonucleotides containing a murine IL-6 C/ EBP binding site, or a NF-kB consensus oligonucleotide. C/EBP probes were labeled with a ATP. NF-kB probes were labeled with c ATP. DNA binding reactions were performed at room temperature in a 25 ml reaction mixture containing 6 ml of nuclear extract and 5 ml of 56 binding buffer Ficoll, 50 mM HEPES pH 7.9, 5 mM EDTA, 5 mM dithiothreitol). The remainder of the reaction mixture contained KCl at a final concentration of 50 mM, Nonidet P-40 at a final concentration of 0.1%, 1 mg of poly, 200 pg of probe, bromphenol blue at a final concentration of 0.06%, and water to final volume of 25 ml. Samples were electrophoresed through 5.5% polyacrylamide gels in 16 TBE at 190 V for approximately 3.5 h, dried under vacuum, and exposed to X-ray film. For supershifts, nuclear extracts were preincubated with antibodies for 0.5 h at 4uC prior to the binding reaction. The following antibodies were purchased from Santa Cruz, CA: NF-kB p50, p52, p65, RelB, cRel, C/EBPa, C/EBPb, C/EBPd, C/EBPe, C/EBPc, and normal rabbit immunoglobulin G. Statistical Analysis All values were expressed as the mean 6 S. E. M. Significance was assigned where p,0.05. Data sets were analyzed using Student’s t test or one-way ANOVA, with individual group means being compared with the Student-Newman-Keuls multiple comparison test. Acknowledgments We greatly appreciate the gift of the expression vectors for C/EBPb, IL-6 promoter-luciferase construct containing a mutated NF-kB binding site provided by Richard C. Schwartz, IL-6 promoter-luciferase construct containing a mutated C/EBP binding site provided by Gail A. Bishop, and 2XC/EBP-luc reporter plasmid provided by Peter Johnson. Electrophoretic Mobility Shift Assay Nuclear extracts of MLE12 cells were prepared as follows. Cells were lysed in 15 mM KCl, 10 mM HEPES, 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, and complete protease inhibitors for 10 min on ice. Nuclei were pelleted by centrifugation at 14,0006 g for 20 sec at 4uC. Proteins were extracted from nuclei by incubation at 4uC with vigorous vortexing in buffer C Alveolar epithelial type II cell: defender of the alveolus revisited. Respir Res 2: 3346. Whitsett JA, Wert SE, Weaver TE Alveolar surfactant homeostasis and the pathogenesis of pulmonary disease. Annu Rev Med 61: 105119. Mason RJ Biology of alveolar type II cells. Respirology 11 Suppl: S1215. Kannan S, Huang H, 345627-80-7 site Seeger D, Audet A, Chen Y, et al. Alveolar epithelial type II cells activate alveolar macrophages and mitigate P. Aeruginosa infection. PLoS One 4: e4891. 5. Vanderbilt JN, Mager EM, Allen L, Sawa T, Wiener-Kronish J, et al. CXC chemokines and their receptors are expressed in type II cells and upregulated following lung injury. Am J Respir Cell Mol Biol 29: 661668. Thorley AJ, Goldstraw P, Young A, Tetley TD Primary human alveolar type II epithelial cell CCL20 induced dendritic cell migration. Am J Respir Cell Mol Biol 32: 262267. Sato K, Tomioka H, Shimizu T, Gonda T, Ota F, et al. Type II alveolar