Onspecific binding sites. Then, the slides were incubated with a goat anti-mouse SPLUNC1 antibody (R D systems, Minneapolis, MN) overnight at 4uC, followed by incubation with biotinylated horse anti-mouse IgG for one hour at room temperature. Thereafter, avidin-biotin-peroxidase complex (Vector Laboratories, AKT inhibitor 2 Burlingame, CA) was added to the slides for 45 min at room temperature. After rinsing the slides in TBS, 0.03 aminoethylcarbazole (AEC) in 0.03 hydrogen peroxide was used as a substrate to develop a peroxide-dependent red color reaction. Nuclear counterstaining was performed by using Mayer’s hemotoxylin (Sigma-Aldrich, St. Louis, MO). The area of SPLUNC1 protein in epithelium of all medium-sized airways (defined as the basement membrane perimeter of 600?00 mm, and maximal diameter/minimal diameter #2, [26]) (n = 4 to 6 airways/mouse) lungs was quantified in a blinded fashion by using the National Institutes of Health Scion Image program (Bethesda, MD). The results were expressed as percentage of airway SPLUNC1 protein area/total airway Pentagastrin epithelial area.ELISA of Mouse KC and IL-KC and IL-6 protein levels in mouse BALF were determined by using mouse KC and IL-6 DuoSet ELISA Development kits (R D Systems, Minneapolis, MN) as per manufacturer’s instruction.Statistical AnalysisNormally distributed data were presented as means 6 SEM and analyzed using the student’s t-test for two group comparison or two-way analysis of variance (ANOVA) for multiple group comparisons. Non-normally distributed data were compared using Wilcoxon rank-sum test. A value of P,0.05 was regarded as statistically significant.AcknowledgmentsWe would like to thank Dr. Yvonne M.W. Janssen-Heininger at University of Vermont (Burlington, VT) for kindly providing us with the conditional NF-kB transgenic mice. We would also like to thank 18325633 Paratek Pharmaceuticals (Boston, MA) for providing us with the tetracycline analog 9-TB.SPLUNC1 Immunohistochemistry (IHC)Because there is no mouse SPLUNC1 ELISA available, we measured airway epithelial SPLUNC1 protein by IHC. Formalinfixed and paraffin-embedded mouse lung sections were deparaffinized, rehydrated, followed by antigen retrieval with microwave boiling in 10 mM citrate buffer (pH 6.0) for 12 min. Sections were treated with 0.3 24195657 hydrogen peroxide in 0.05 M Tris buffered saline (TBS, pH 7.6) for 30 min to inhibit endogenous peroxidase, followed by incubation with 10 normal rabbit serumAuthor ContributionsConceived and designed the experiments: DJ FG HWC. Performed the experiments: DJ FG SS QW MM SC JT HWC. Analyzed the data: DJ FG SS QW MM SC JT HWC. Contributed reagents/materials/analysis tools: DJ MLN FG SS QW MM SC JT HWC. Wrote the paper: DJ.
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) serves as the main gateway for inorganic carbon to enter metabolic pathways in most ecosystems and hence is unique in its importance to support life. Observations of significant variation in Rubisco kinetics between plant species [1,2,3], the correlation of Rubisco kinetics with temperature [4] and CO2 availability [5], and positive selection on Rubisco at the molecular level in all principal lineages of land plants [6] support the hypothesis that all Rubiscos may be well adapted to their subcellular environment [7]. However, the molecular mechanisms responsible for optimizing the relationship between Rubisco specificity and its maximum rate of catalytic turnover in particular conditions are still open to debate [.Onspecific binding sites. Then, the slides were incubated with a goat anti-mouse SPLUNC1 antibody (R D systems, Minneapolis, MN) overnight at 4uC, followed by incubation with biotinylated horse anti-mouse IgG for one hour at room temperature. Thereafter, avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA) was added to the slides for 45 min at room temperature. After rinsing the slides in TBS, 0.03 aminoethylcarbazole (AEC) in 0.03 hydrogen peroxide was used as a substrate to develop a peroxide-dependent red color reaction. Nuclear counterstaining was performed by using Mayer’s hemotoxylin (Sigma-Aldrich, St. Louis, MO). The area of SPLUNC1 protein in epithelium of all medium-sized airways (defined as the basement membrane perimeter of 600?00 mm, and maximal diameter/minimal diameter #2, [26]) (n = 4 to 6 airways/mouse) lungs was quantified in a blinded fashion by using the National Institutes of Health Scion Image program (Bethesda, MD). The results were expressed as percentage of airway SPLUNC1 protein area/total airway epithelial area.ELISA of Mouse KC and IL-KC and IL-6 protein levels in mouse BALF were determined by using mouse KC and IL-6 DuoSet ELISA Development kits (R D Systems, Minneapolis, MN) as per manufacturer’s instruction.Statistical AnalysisNormally distributed data were presented as means 6 SEM and analyzed using the student’s t-test for two group comparison or two-way analysis of variance (ANOVA) for multiple group comparisons. Non-normally distributed data were compared using Wilcoxon rank-sum test. A value of P,0.05 was regarded as statistically significant.AcknowledgmentsWe would like to thank Dr. Yvonne M.W. Janssen-Heininger at University of Vermont (Burlington, VT) for kindly providing us with the conditional NF-kB transgenic mice. We would also like to thank 18325633 Paratek Pharmaceuticals (Boston, MA) for providing us with the tetracycline analog 9-TB.SPLUNC1 Immunohistochemistry (IHC)Because there is no mouse SPLUNC1 ELISA available, we measured airway epithelial SPLUNC1 protein by IHC. Formalinfixed and paraffin-embedded mouse lung sections were deparaffinized, rehydrated, followed by antigen retrieval with microwave boiling in 10 mM citrate buffer (pH 6.0) for 12 min. Sections were treated with 0.3 24195657 hydrogen peroxide in 0.05 M Tris buffered saline (TBS, pH 7.6) for 30 min to inhibit endogenous peroxidase, followed by incubation with 10 normal rabbit serumAuthor ContributionsConceived and designed the experiments: DJ FG HWC. Performed the experiments: DJ FG SS QW MM SC JT HWC. Analyzed the data: DJ FG SS QW MM SC JT HWC. Contributed reagents/materials/analysis tools: DJ MLN FG SS QW MM SC JT HWC. Wrote the paper: DJ.
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) serves as the main gateway for inorganic carbon to enter metabolic pathways in most ecosystems and hence is unique in its importance to support life. Observations of significant variation in Rubisco kinetics between plant species [1,2,3], the correlation of Rubisco kinetics with temperature [4] and CO2 availability [5], and positive selection on Rubisco at the molecular level in all principal lineages of land plants [6] support the hypothesis that all Rubiscos may be well adapted to their subcellular environment [7]. However, the molecular mechanisms responsible for optimizing the relationship between Rubisco specificity and its maximum rate of catalytic turnover in particular conditions are still open to debate [.
