Mice infected with PA103 also exhibited increased inflammatory markers (Fig. CA). The gel extraction kit and QIAprep spin miniprep kits were from Qiagen (Valencia, CA). FuGene6 transfection reagent was purchased from Roche Diagnostics (Indianapolis, IN). Nucleofector transfection kits were from Amaxa (Gaithersburg, MD). Immobilized protein A/G beads were from Pierce (Rockford, IL). All DNA sequencing was performed by the University of Iowa DNA Core Facility. Cell culture. MLE cells were cultured in Dulbecco’s altered Eagle mediumCF-12 (Gibco) supplemented with 2 or 10% fetal bovine serum (DMEM-2 or -10). In some studies, cells were serum starved (DMEMCF-12) and infected with PA103 at a multiplicity of contamination (MOI) of 10 for 1 h or treated with “type”:”entrez-nucleotide”,”attrs”:”text”:”A23187″,”term_id”:”833253″A23187 at 10 nM for 4 h. In other experiments, cells were incubated with 20 mM NH4Cl, 1:1,000 leupeptin, or a 1:1,000 dilution of lactacystin for 24 h. Cell lysates were prepared by brief sonication in 150 mM NaCl, 50 mM Tris, 1.0 mM EDTA, 2 mM dithiothreitol (DTT), 0.025% sodium azide, and 1 mM phenylmethylsulfonyl fluoride (buffer A) at 4C. Expression of recombinant proteins and RNA inhibition (RNAi). Cellular expression of plasmids was facilitated using the Amaxa nucleofector system, with transfection efficiencies of 90% (3). MLE cells (4 106) were plated in 100-mm dishes for 24 h, infected with an adenovirus-CaM (Ad-CaM) vector or an empty vector (Ad-Con) at an MOI of 40 for 12 h, followed by transfection with FBXL2 plasmid. For cameleon expression, 5 104 cells were plated in 96-well plates. In baculovirus studies, 2 105 cells were plated in 35-mm glass-bottom dishes for 24 h and then infected with baculovirus-cameleon following the manufacturer’s instructions. Recombinant CCT was expressed and purified as described previously (26). For siRNA studies, 1 106 cells were transfected using WIKI4 nucleofection with 0.2 nmol of scrambled RNA or FBXL2 siRNA and harvested after an additional 48 h. Bacterial culture. PA103 and PA103 mutants were kindly provided by Tim Yahr (University of Iowa, Iowa City, IA). Inocula were freshly prepared prior to experiments from frozen stocks of PA103 (frozen at mid-log phase; optical density at 540 nm of 0.8). PA103 was maintained in Vogel-Bonner minimal agar. Cultures were plated and produced overnight from frozen stock. Overnight plate cultures were then inoculated in tryptic soy broth supplemented with 1% glycerol and 100 mM sodium glutamate (TSB++) and produced by rotary shaking at 37C to log phase (3). Animal studies. Male C57LB/6 mice (purchased from Jackson Laboratories) were acclimated at the University of Iowa Animal Care Facility and maintained according to all federal and institutional animal care guidelines and under a University of Iowa Institutional Animal Care and Use Committee WIKI4 (IACUC)-approved protocol. Mice were deeply anesthetized with ketamine (80 to 100 mg/kg of body weight, intraperitoneally [i.p.]) and xylazine (10 mg/kg, i.p.), and then the larynx was well visualized under a fiber optic light source before endotracheal intubation with a 3/400 24-gauge plastic catheter. Replication-deficient adenovirus (Ad5) alone or Adv-CaM (109 PFU in 50 l of 10 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.1% bovine serum albumin) was instilled intratracheally (i.t.) on day 1, after which animals were allowed to recover for 48 h. Following recovery, mice were deeply anesthetized again, followed by administration of (PA103; 107 CFU/mouse, i.t.) for 1 h. A tracheostomy was performed, and a metal 1.2-mm (internal diameter) tracheal cannula was inserted and tied firmly into place. An electrocardiograph tracing was monitored to ascertain any adverse effects of the ventilatory maneuvers. The mice were deeply anesthetized, paralyzed, and mechanically ventilated WIKI4 with a positive end expiratory pressure (PEEP) of 3, and a quasistatic volume pressure determination was performed by using a FlexiVent system (4). Lavage fluids were collected from mice to isolate surfactant, as described previously (20). Immunoblot analysis. Equal amounts of Mouse monoclonal to GABPA total protein in sample buffer were resolved by SDS-PAGE and transferred to nitrocellulose, and immunoreactive proteins were detected as described previously (4). The dilution factor for primary and secondary antibodies was 1:2,000. CCT was purified to homogeneity from rat WIKI4 liver as described previously (4). Coimmunoprecipitation. Total cellular protein or the.
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