Expression of mast cell proteases in mast cells derived from B6, B6-cma, and DBA/2 mice.

<p>Expression of indicated mast cell proteases together with house-keeping gene GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was analyzed by real time PCR using specific PCR primers shown in the top panel. Data represent relative gene expression levels calculated based on threshold cycles and standard curves obtained with serial dilutions of purified PCR products. Error bars denote standard deviation (n≥3). *p<0.0001 in comparison with B6 control mice.</p

Association of Mcp-2 and Mcp-4 overexpressions in BMMCs with gene variants.

<p><b>A.</b> Mice were genotyped for the Mcp-2 gene variant by allele-specific PCR and the Mcp-4 gene variant by restriction fragment length polymorphism (RFLP) with NdeI. BMMCs derived from these mice were analyzed for Mcp-2 and Mcp-4 protein expressions by Commassie blue staining and western blotting. <b>B.</b> BMMCs from B6 and B6-cma mice were analyzed for chymase activity. In at least 45 mice analyzed, there is a perfect correlation of Mcp-2 and Mcp-4 gene variants with overexpression of Mcp-2 and Mcp-4 and increased chymase activity.</p

Comparison of Mcp-2 and Mcp-4 protein levels in mast cells from B6, B6-cma, and DBA/2 mice.

<p>Mast cells were derived from bone marrow (BM) and peritoneal cavity (PC) of B6, B6-cma, and DBA/2 mice. <b>A.</b> Cell extracts were resolved on 12.5% SDS gels followed by Coomassie blue staining (top panel) or western blotting with indicated antibodies. <b>B.</b> Cells were subjected to Wright-Giemsa staining (top panel) or immunofluorescent staining with indicated antibodies.</p

Chymase activity in mast cells from B6, B6-cma, and DBA/2 mice.

<p>Mast cells were derived from bone marrow (BM) and peritoneal cavity (PC) of B6, B6-cma, and DBA/2 mice. Cells were either extracted for assays of total chymase activity (A) or treated with antigen to induce degranulation for determination of secreted chymase activity (B). Specific activity was calculated in reference to total proteins in cell pellets. Error bars denote standard deviation (n≥3). *p<0.0001 in comparison with B6 control mice.</p

Reporter gene assays.

<p><b>A.</b> Schematic diagram of report gene constructs. SNPs are indicated, and black blocks represent CANNTG motifs (E-boxes). <b>B.</b> Report gene constructs were transfected into mast cells derived from bone marrow of B6 mice. <b>C.</b> Report gene constructs together with pcDNA3 plain vector or pcDNA3-Mitf-A were transfected into NIH3T3 cells. Relative report gene expression is represented by firefly luciferase activity normalized against that of renilla luciferase. Error bars denote standard deviation (n≥3).</p