Figure 1.

Characterization of Pro by Western Blot and use in flow cytometry. (A)Human lung fibroblast culture supernatant was Western blotted using Pro (0.2 μg/ml) competed with Non-specific peptide (immunogenic peptide for Telo, 0.4 μg/ml = 142-fold molar excess), with Specific peptide (immunogenic peptide for Pro, 0.4 μg/ml = 142-fold molar excess), or with serial 5-fold dilutions of Specific peptide. The prominent polypeptide detected is full-length 180 kD procollagen Iα1. The minor, slightly smaller polypeptide lacks the N-terminal propeptide. (B) Total lung cells from a bleomycin-treated mouse sacrificed 28 days after initiation of treatment were immunolabeled for CD45 and, as indicated, with no additional antibody, with Pro competed with specific peptide (142-fold molar excess), and with Pro competed with non-specific peptide (142-fold molar excess). Scatter plots are presented with CD45 fluorescence intensity on the y-axis and Pro (or unlabeled) fluorescence intensity on the x -axis. (C) Total lung cells from a saline-treated mouse and a bleomycin-treated mouse sacrificed 28 days after initiation of treatment were immunolabeled for CD45 and Pro. Scatter plots are presented with CD45 fluorescence intensity on the y-axis and Pro fluorescence intensity on the x-axis. The zones named Regions I–IV are shown and are used throughout this study. (D) CD45 or Pro levels in Regions I–IV, and I + II are shown in terms of the average ± s.e.m. of the median fluorescence intensity of each of 18 saline and 26 bleomycin mice sacrificed 28 days after initiation of treatment. S, saline; B, bleomycin. Error bars are not shown and the t-test was not applied to these data because CD45 and Pro fluorescence intensity were the criteria used to define Regions I–IV.