Figure 4

Increased expression of PA200 in primary human bronchial basal cells and TGF-β1 activated myofibroblasts. (A) PA200 (PSME4) mRNA levels analyzed in primary human bronchial basal cells at day 0 (d0) and day 28 (d28) of differentiation into bronchial epithelial cells. RPL19 served as housekeeping gene (one-sample t-test, primary human bronchial basal cells from n = 3 different patients). (b) Analysis of PA200 protein expression in cells analyzed in (a). Amidoblack staining served as a loading control for Western blotting (one-sample t-test, primary human bronchial basal cells from n = 3 different patients). (c) Primary human lung fibroblasts (phLF) treated with TGF-β1 for 48 h analyzed for myofibroblast markers collagen1α1 (COL1A1), fibronectin (FN) and α-smooth muscle actin (αSMA) protein expression with densitometric analysis of protein levels relative to housekeeping protein β-Actin normalized to controls (one-sample t-test, in phLF from n = 6 organ donors). (d) Cells analyzed in (c) were assessed for PA200 (PSME4), 19S regulator subunit RPT5 (PSMC3) and 20S proteasome subunits α7 (PSMA3) and β5 (PSMB5) mRNA expression. RPL19 served as housekeeping gene (one-sample t-test, in phLF from n = 6 organ donors). (e) Protein expression of PA200, RPT5, α1-7 and β5 in cell extracts analyzed in (c) with densitometric analysis of protein levels relative to housekeeping protein β-Actin normalized to controls (one-sample t-test, in phLF from n = 6 organ donors). (f) Native gel analysis of native extracts of TGF-β1-treated phLF with substrate overlay for the chymotrypsin-like (CT-L) proteasome activity and immunoblotting for PA200. Figure shows representative results of experiments performed in phLF from n = 3 organ donors. Full-length immunoblots are presented in Supplementary Fig. S5.