Inhibition of collagen I and ASMA expression and MEK/ERK activation in fibroblasts by CSD and its subdomains. Serum-starved NLF and SLF were incubated for an additional 6 h in fresh serum-free medium containing 5 μM of either CSD, Control peptide, or the indicated subdomain of CSD. The Antennapedia Internalization Sequence alone was routinely used as the Control peptide; when tested scrambled CSD attached to the Antennapedia Internalization Sequence gave similar results. The levels of collagen I in the culture medium and of pMEK, MEK, pERK, ERK, ASMA, and actin (loading control) in the cell layer were determined by Western blotting as described in the Methods (A). (B) Densitometric quantification combining data from three experiments similar to A performed with three independent pairs of NLF and SLF. For comparisons of CSD and related peptides with Control peptide*** indicates p < 0.001, ** indicates p < 0.01, and * indicates p < 0.05. For NLF/Control peptide vs SLF/Control peptide for each chart in (B) p < 0.01. Immunofluorescent detection of effects of CSD and its subdomains on pERK (C) and ASMA (D) expression in NLF and SLF. Cells were cultured on coverslips under the conditions described above, then fixed and stained to detect the indicated proteins. Nuclei were counterstained with DAPI.