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Cleavage of PDGF Receptor on Periodontal Ligament Cells by Elastase

Division of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan;

View larger version (27K): [in a new window]   Figure 1. PDGFR expression on PDL cells after elastase treatment. (A,B) PDL cells were collected from confluent monolayers with the use of Cell Dissociation Solution®. (C,D) Cells were treated with the indicated concentrations of elastase for 30 min at 37°C, or (E,F) were treated with 5 µg/mL elastase for the indicated time. Expressions of PDGFR- and -ß on the cell surface were assessed by flow cytometry, as described in MATERIALS & METHODS. An isotype-matched antibody was used as the negative control (broken line). Findings (A,B) are representative of 3 independent experiments. The results (C-F) are shown as MFI ± SE of duplicate assays, and statistical significance is shown (*P < 0.05 vs. control).

View larger version (40K): [in a new window]   Figure 2. Degradation of PDGFR by elastase treatment. (A) PDL cells collected from the confluent monolayer were treated with the indicated concentration of elastase for 30 min, as described in MATERIALS & METHODS. (B) Cells collected from confluent monolayers were fixed with 1% (w/v) paraformaldehyde for 5 min at room temperature. After being washed, unfixed and fixed cells were incubated with 5 µg/mL elastase for 30 min. Elastase was pre-treated with 1 mmol/L of HLE/CMK for 15 min at 37°C before use. (C) Confluent monolayer cells were treated with 5 µg/mL elastase for 30 min. Cell lysates (A,B,C) were prepared as described in MATERIALS & METHODS. (D) Human rPDGFR- and -ß were treated with the indicated molar ratio of elastase for 30 min. Samples were subjected to 7.5% (A,B,C, and lower D) or 10% (upper D) SDS-PAGE, and transferred to a PVDF membrane. The blot was probed with an anti-PDGFR- and PDGFR-ß polyclonal Ab. Molecular mass markers (kDa) are shown on the right. Representative findings of 3 independent experiments are shown.

View larger version (44K): [in a new window]   Figure 3. Inhibition of PDGF-triggered MAPK activation by elastase treatment. Confluent monolayer cells were starved of FBS for 24 hrs in -MEM, and then treated with 5 µg/mL elastase for 30 min, followed by stimulation with 50 ng/mL of PDGF-AA or PDGF-BB for 5 min. Cell lysates were analyzed by Western blotting with phospho-specific p44/42 ERK1/2 (Thr202/Tyr204), SAPK/JNK (Thr183/Tyr185), and p38 MAPK (Thr180/Tyr182) antibodies for detection of the phosphorylation of MAPK. Antibodies against total p44/42 ERK1/2, SAPK/JNK, and p38 MAPK were used as controls. Molecular mass markers (kDa) of MAPK are shown on the right: ERK1, 42 kDa; ERK2, 44 kDa; JNK-1, 46 kDa; JNK-2, 54 kDa; and p38, 43 kDa. Representative findings of 3 independent experiments are shown.