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Fig. 4. CD44 mediates the activation of TGFβ in murine adult fibroblasts in response to wounding. CD44KO fibroblasts generate reduced levels of active TGFβ in response to wounding compared with CD44WT fibroblasts. Levels of total and active TGFβ in conditioned media from unwounded and wounded CD44WT and CD44KO fibroblasts were determined after 24 hours, in the presence or absence of anti-TGFβ antibody (5 µg/ml). Values were normalized to total protein concentration. Cell viability was comparable between CD44WT and CD44KO fibroblasts under each condition. (A) Levels of active (top) and total (bottom) TGFβ (pg/ml) produced in conditioned media under each condition, as measured in three independent experiments. Within each experiment, the conditions were done in triplicate and also assayed in triplicate. Error bars represent s.e.m. (B) CD44KO fibroblasts exhibit lower levels of nuclear Smad2-P (pSmad2). Immunoblot analysis and corresponding quantification of Smad2-P of nuclear lysates from CD44WT and CD44KO fibroblasts unwounded, wounded and treated with active TGFβ1 (0.02 nM), extracted after 1 hour. Immunoblot analysis was done with anti-Smad2-P (Ser465/467) and anti-Rb (loading control). Immunoblot is representative of one experiment performed three times with similar results. Corresponding quantification is an average of the three experiments normalized to the loading control. Error bars represent s.e.m.
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