FIG. 2.
Pcolce−/− MEFs are devoid of PCOLCE1 and have reduced processing of the C propeptides of procollagen types I and V. (A) Twenty-five nanograms each of recombinant PCOLCE1 and -2 is compared by Western blotting to samples corresponding to 30 μl medium and 10% of the cell layer of 10-ml-medium/10-cm-culture-dish cultures of Pcolce+/+, Pcolce+/−, and Pcolce−/− MEFs. (B) An autofluorogram of 24-h-[3H]proline-radiolabeled material shows type I procollagen processing to be diminished in Pcolce−/− MEF medium, compared to Pcolce+/+ and Pcolce+/− MEF media. (C) An autofluorogram of pulse (1-h)/chase (9-h) [3H]proline-radiolabeled MEF medium samples, run on a long SDS-polyacrylamide gel to separate processing intermediates, shows increased and decreased amounts of pCα1(I) and pNα1(I), respectively, in a Pcolce−/− sample compared to wild type (+/+). (D and E) Western blotting shows that processing of pNα1(V) to mature α1(V) chains is unaffected (D) but that processing of pro-α2(V) to pNα2(V) chains is decreased (E, left panel) in Pcolce−/− MEFs, compared to wild type (+/+). The Western blot in the right panel of panel E compares processing of type I procollagen in the same samples used for analyzing type V procollagen processing in the left panel of panel E and in panel D.