Figure 3
TGF-β 1 stimulated α-SMA and FN1-EDA protein expression were substantially reduced by forskolin in CT-, PF- and DC-derived fibroblasts. CT-, PF- and DC-derived fibroblasts (a, b, c) grown on 6-well culture dishes were stimulated with forskolin (10 μM), TGF-β1 (2 ng/ml), with both agents, or were left untreated for 24 hours in MEM-α medium containing 0.1% dialyzed FBS. Whole cell lysates were collected after 24 hours and the samples were processed for Western immunoblotting with specific antibodies for α-SMA and FN1-EDA (20 μg/lane). Specificity of the modulation and identical protein loading was confirmed with a loading control GAPDH antibody. Densitometry analysis was done on protein bands obtained from three independent experiments performed in triplicate using two independent primary cultures of CT-, PF- and DC-derived fibroblasts (Figure 3d and 3e). A representative immunoblot is shown here.