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Analysis of mRNA of mast cell proteases and activity of tryptase and carboxypeptidase A in BMMCs. BMMCs were derived from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice for 6 weeks. A, total RNA was prepared from BMMCs and used for analysis of the expression of mouse mast cell protease-1 (mMCP-1), mMCP-2, mMCP-4, mMCP-5, mMCP-6, mMCP-7, carboxypeptidase A, serglycin, and β-actin. B, tryptase activity of the extracts from BMMCs was determined as described under “Experimental Procedures.” Each bar represents the mean ± S.D. from three culture dishes prepared simultaneously. Similar results were obtained from the three independent experiments. *, p < 0.001; **, p < 0.0001. C, protein extracts from the BMMCs were subjected to 10% SDS-PAGE. Western blots were probed using an antibody against mMCP-6 or actin followed by ECL as described under “Experimental Procedures.” D, carboxypeptidase activity of the extracts from BMMCs was determined as described under “Experimental Procedures.” Each bar represents the mean ± S.D. from three culture dishes prepared simultaneously. Similar results were obtained from another independent experiment. *, p < 0.01; **, p < 0.001.

Journal: The Journal of Biological Chemistry

Article Title: Mice Deficient in N -Acetylgalactosamine 4-Sulfate 6- O -Sulfotransferase Are Unable to Synthesize Chondroitin/Dermatan Sulfate containing N -Acetylgalactosamine 4,6-Bissulfate Residues and Exhibit Decreased Protease Activity in Bone Marrow-derived Mast Cells *

doi: 10.1074/jbc.M109.084749

Figure Lengend Snippet: Analysis of mRNA of mast cell proteases and activity of tryptase and carboxypeptidase A in BMMCs. BMMCs were derived from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice for 6 weeks. A, total RNA was prepared from BMMCs and used for analysis of the expression of mouse mast cell protease-1 (mMCP-1), mMCP-2, mMCP-4, mMCP-5, mMCP-6, mMCP-7, carboxypeptidase A, serglycin, and β-actin. B, tryptase activity of the extracts from BMMCs was determined as described under “Experimental Procedures.” Each bar represents the mean ± S.D. from three culture dishes prepared simultaneously. Similar results were obtained from the three independent experiments. *, p < 0.001; **, p < 0.0001. C, protein extracts from the BMMCs were subjected to 10% SDS-PAGE. Western blots were probed using an antibody against mMCP-6 or actin followed by ECL as described under “Experimental Procedures.” D, carboxypeptidase activity of the extracts from BMMCs was determined as described under “Experimental Procedures.” Each bar represents the mean ± S.D. from three culture dishes prepared simultaneously. Similar results were obtained from another independent experiment. *, p < 0.01; **, p < 0.001.

Article Snippet: DOCOSIL (4.6 × 150 mm) was from Senshu Scientific Co. Ltd., Tokyo; Geneticin was from Invitrogen; Nanosep Centrifugal Devices 3K was from Nihon Pall Ltd, Tokyo; WEHI-3 cells were from American Type Culture Collection; NIH-3T3 cells were from RIKEN Cell Bank, Tsukuba, Japan; stem cell factor (SCF) was from PeproTech, Inc., Rocky Hill, NJ; antibody against mMCP-6 (sc-32473) was from Santa Cruz Biotechnology, Inc., Santa Cruz, CA; anti-actin antibody was from Sigma; S-2586 for chymotrypsin-like proteases and S-2288 for trypsin-like proteases were from Chromogenix, Milano, Italy; M-2245 for carboxypeptidase A was from Bachem AG, Bubendorf, Switzerland.

Techniques: Activity Assay, Derivative Assay, Mutagenesis, Expressing, SDS Page, Western Blot