Figure 5.
Effects of Smad3 siRNA on activities of MAP kinases, NF-κB/PAI-1, and expression of cytokines in BMMCs activated with antigen/antibody reaction. Smad3 siRNA-expressing vectors were generated in BMMCs using the Silencer Express Kit. Transfected BMMCs (1 × 106 cells) were activated with 0.1 μg/ml anti-DNP IgE antibody and DNP-HSA (1.0 μg/ml) for 6 h. CSE solution (OD 2.0, 1.0%) was treated during DNP-HAS stimulation, as described in "Materials and Methods". Expression or activity of Smad3, MAP kinases, transcription factors and cytokines were determined in extract protein and nuclear protein extracts prepared from BMMCs using western blot and RT-PCR, respectively. Mean ± SEM (n = 4) for values measured by densitometry analysis was indicated by histogram. (A) Band images and histogram for protein and activity of Smad3. (B) Band images and histogram for expressions of NF-κB and PAI-1. (C) Band images and histogram for activities of MAP kinases. (D) Band images and histogram for cytokine mRNA expressions. BM, BMMCs; Ag/Ab Rx, anti-DNP IgE antibody/DNP-HSA reaction; -, +, without and with Smad3 siRNA. ***, P < 0.001versus control (BMMCs alone). +++, P < 0.001 versus CSE-treated/activated-BMMCs.