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Tropoelastin Expression by 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 (34K): [in this window] [in a new window]   Figure 1. Tropoelastin mRNA expression by HGF and HPLF. (A) Northern blotting analysis. HGF and HPLF were harvested at different times, as indicated. Total cell RNA was prepared, and 2 µg was analyzed by Northern blotting, as described in MATERIALS & METHODS. The top panel shows a representative Northern blot probed for tropoelastin mRNA expression, after which the blot was stripped and re-probed for β-actin mRNA expression (bottom panel). (B) The bands in (A) were analyzed densitometrically, and the HGF tropoelastin mRNA to β-actin mRNA ratios are plotted on a bar graph. The results are expressed as percentages relative to the four-week value and represent the means ± SD (standard deviation) of three independent experimental determinations. (C) Photograph of ethidium-bromide-stained 1% agarose electrophoresis gels showing the RT-PCR products of mRNAs extracted from HGF and HPLF after culture for 4 wks. A tropoelastin band from the HGF sample appearing at 1100-bp sites is present in lane 1. No detectable tropoelastin band was obtained from HPLF (lane 3). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) bands from HGF and HPLF samples appeared at 998-bp sites in lanes 2 and 4, respectively. The molecular mass standards (lane STD) were Lambda DNA/(EcoRI and HindIII) fragments.

View larger version (68K): [in this window] [in a new window]   Figure 2. Immunoblotting of tropoelastin (upper panel). HGF and HPLF underwent lysis at the indicated times, and equal amounts of protein (100 µg) were separated by SDS-PAGE and transferred to PVDF membranes. The blots were probed with an anti-tropoelastin antibody and developed with ECL reagent. To ensure that equivalent amounts of extracted proteins were present in each lane, we stained another PVDF membrane with Amido Black 10B reagent before hybridization (lower panel). The results are representative of three independent experiments.

Journal of Dental Research, Vol. 81, No. 3, 198-202 (2002)
DOI: 10.1177/154405910208100311


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