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Gene Expression and Accumulation of Fibrillin-1, Fibrillin-2, and Tropoelastin in Cultured Periodontal Fibroblasts

Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan;

View larger version (31K): [in a new window]   Figure 1. Fibrillin-1, fibrillin-2, and tropoelastin mRNA expression in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPLF). (A) Northern blot analysis. HGF and HPLF were harvested at the time-points indicated. Total cell RNA was extracted, and 1 µg was analyzed by Northern blotting analysis as described in MATERIALS & METHODS. (B) Densitometric analysis of the time-course of changes in the levels of expression of fibrillin-1, fibrillin-2, and tropoelastin mRNA, measured as shown in (A). All mRNA expression levels were analyzed with the use of National Institutes of Health imaging software and were normalized relative to ß-actin mRNA expression. The levels of expression in the HGF two-week culture samples were arbitrarily assigned the value of 1. The results represent the means + SD (standard deviation) of three independent experimental determinations.

View larger version (46K): [in a new window]   Figure 2. Immunodetection of fibrillin-1, fibrillin-2, and tropoelastin in matrix samples from HGF and HPLF cell layers after 2, 4, and 6 wks of culture. Equal amounts of matrix samples (100 µg) that had been harvested from HGF and HPLF cell layers after 2, 4, and 6 wks of culture were separated by SDS-PAGE and transferred to Immobilon-P membranes. The blots were probed with anti-fibrillin-1 (A), anti-fibrillin-2 (B), and anti-tropoelastin (C) antibodies. To ensure that equivalent amounts of the extracted proteins were present in each lane, we stained another membrane with Amido Black 10B reagent before hybridization (lower panel in A, B, and C). The results are representative of the three independent experiments. The positions of the size markers are indicated on the right. Densitometric scanning of the bands was performed with the use of National Institutes of Health imaging software.