|ONLINE SUPPLEMENTARY INFORMATION
|Year : 2015 | Volume
| Issue : 3 | Page : 120-121
Phosphodiesterase 4B is essential for lipopolysaccharide-induced CC chemokine ligand 3 production in mouse macrophages
|Date of Web Publication||15-Jun-2015|
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
. Phosphodiesterase 4B is essential for lipopolysaccharide-induced CC chemokine ligand 3 production in mouse macrophages. J Med Sci 2015;35:120-1
| Materials and Methods|| |
Protein sample preparation and two-dimensional gel electrophoresis
Proteins in the medium supernatants were precipitated in trichloroacetic acid (10%), followed by washing three times with ice-cold acetone containing 0.07% β-mercaptoethanol. The proteins were air-dried and resuspended in two-dimensional (2D) sample buffer (9 M urea, 0.5% Triton X-100, 65 mM DTT, and 2% pharmalyte pH 3-10). Protein concentration was determined using 2D Quant Kit (GE Healthcare, NJ, USA).
Total protein (200 μg) in 2D sample buffer (100 μl) was mixed with 150 μl of rehydration buffer (9 M urea, 0.5% Triton X-100, 13 mM DTT, 0.5% pharmalyte pH 3-10, and 0.01% bromophenol blue) before loading onto 13-cm pH 3-10 immobilized gradient strip for the first dimensional separation. Isoelectric focusing (IEF) was performed on Ettan IPGphor III horizontal electrophoresis unit (GE Healthcare, NJ, USA) at 20°C. The strips were rehydrated for 10 h, and then IEF was performed by the following steps: 100 V for 3 h, 500 V for 1 h, 1000 V for 1h, 2000 V for 1h, and a final step of 8000 V for 36,000 Vh. After focusing, IPG strips were equilibrated for 15 min in 50mM Tris-HCl (pH 8.8) containing 6 M urea, 30% glycerol, 2% SDS, and 1% DTT, followed by additional 15 min soaking in 50 mM Tris-HCl (pH 8.8) containing 6 M urea, 30% glycerol, 2% SDS, 2.5% iodoacetamide, and 0.01% bromophenol blue. The second dimension was carried out on 15% SDS-PAGE gels. Electrophoresis was conducted at a constant voltage of 250 V for 8 h in a SE600 Chroma Standard 2D Gel Electrophoresis system (Hoefer, MA, USA). The gels then were silver stained.  Gel images were saved as tif format and analyzed with Delta2D software (Decodon, Germany).
Protein spots were excised from the gel and destained in a solution of 50 mM sodium thiosulfate and 15 mM potassium ferricyanide till the color disappears. After washing with 25 mM ammonium bicarbonate for 10 min, the destained gel pieces were dehydrated in 100% acetonitrile for 5-10 min. Trypsin (12.5 ng/μl) in 50 mM ammonium bicarbonate was added to the gel and sit at 0°C for 30 min to allow the gel to absorb the solution. After removing the excessive trypsin solution, 6-9 μl of 25 mM ammonium bicarbonate was added and digestion was carried out at 37°C for 12-16 h. The peptides were extracted by adding 2-3 μl of 5% formic acid/95% acetonitrile followed by sonication (Delta Sonicator DC400, Taiwan) for 15 min, and the same procedure was repeated three times. The extracted proteins were collected, dried, and stored at −20°C for the following protein identification.
Protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry
The tryptic peptides were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry on an Ultraflex instrument (Bruker Daltonics, MA, USA) for protein identification. The spectra were searched with MASCOT (http://www.matrixscience.com) against the Swiss-Prot database with the following parameters: Trypsin autolysis products, up to one missed cleavage, fixed carbamidomethylation of cysteine and variable oxidation of methionine, peptide tolerance of 50 ppm, and peptide charge of +1. No restrictions on protein molecular weight and pI value were applied. Proteins with a MOWSE score over the threshold 55 for P < 0.05 calculated for the used settings were considered as identified.
Protein identification by N-terminal sequencing
Protein samples prepared from the culture medium were undergone separation by the first-dimensional IEF and the second-dimensional SDS-PAGE as described above. The proteins on the gel were transferred onto the polyvinylidene difluoride membrane using 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid buffer, pH 11, containing 10 % methanol. After staining the membrane with coomassie blue, the spots were excised and the N-terminal sequence of the proteins determined using an automated Edman degradation method with a Perkin Elmer Applied Biosystems Model 494 Procise protein sequencer (Foster City, CA, USA).
Quantitative polymerase chain reaction
Following reverse transcription, the cDNA was used as template for amplification of CC chemokine ligand 3 (CCL3) and GAPDH sequences using the SYBR FAST quantitative polymerase chain reaction (PCR) master mix (KAPA Biosystems, MA, USA) following the manufacturer's protocol. Oligonucleotide primers were as follows: CCL3, 5′-CCAAGTCTTCTCAGCGCCAT-3′ and 5′-TCCGGCTGTAGGAGAAGCAG-3′; GAPDH, 5′-GGAGCGAGACCCCACTAACA-3′ and 5′-ACATACTCAGCACCGGCCTC-3′. The thermal cycler protocol consisted of a 2-min period at 50°C (uracil removal for PCR carryover protection), a 10-min period at 95°C (denaturation of native DNA), and 40 cycles of 15 s at 95°C (denaturation of PCR product) and 1 min at 60°C (annealing/extension). As controls for DNA contamination, total RNA and water were taken as templates. Target gene expression was calculated by the comparative ΔΔ cycle threshold (C t) method for relative quantification after normalization to housekeeping gene (Gapdh) expression. 
Splenic T-cell preparation and chemotaxis assay
Spleen was isolated from C57Bl/6 mice and splenic T cells were purified by depleting B cells using the plates coated with goat anti-mouse IgG + IgM (H + L) (Jackson ImmunoResearch Laboratories, PA, USA),  followed by culturing the remaining cells in RPMI 1640 complete medium for 2 h. The nonadherent cells enriched in T cells were harvested for chemotaxis assay.
T-cell migration was performed in 24-transwell plates with 3-μm pore size polycarbonate filters (Corning, NY, USA) using RPMI 1640 medium supplemented with 1% FBS. Splenic T cells (1 × 10 6) in 100 μl of medium were placed in the upper chamber, and increasing concentrations of recombinant murine CCL3 (PeproTech, NJ, USA) were added in the lower chamber. After 2-h incubation at 37°C, the suspension of cells in the lower chamber was transferred to tubes. The cells were washed with PBS containing 2% FBS followed by fixation in intracellular fixation buffer (eBioscience, CA, USA) overnight. After washing with PBS/2%FBS, 40,000 polystyrene beads (Polysciences, PA, USA) were added to each tube. The cells and beads were counted using FACSCanto II flow cytometer (BD Biosciences, CA, USA), and the migrated cells were calculated as described previously.  To assess the effect of PDE4 inhibition on chemotaxis, splenic T cells were treated with vehicle (DMSO) or rolipram (10 μM) for 20-30 min before transferring to the upper chamber.
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