FIGURE 5.

Analysis of C-P4H-I and C-P4H-II protein expression in control and Hif-1a (A), Hif-2a (B), and Vhl (C) floxed chondrocytes. The Hif-1a^f/f, Hif-2a^f/f, and Vhl^f/f chondrocytes were cultured in a monolayer and transduced on day 1 after plating with adenoviruses producing either β-galactosidase or Cre recombinase. On day 10 the cells were exposed to 1% O₂ for 8 or 24 h, or the culture was continued in normoxia. Total cell lysates were analyzed by 8% nondenaturing PAGE followed by Western blotting with antibodies against the C-P4H α(I) and α(II) subunits. The accumulation of HIF-1α (A and C) and HIF-2α (B) was studied by 8% SDS-PAGE followed by Western blotting with an antibody recognizing HIF-1α or HIF-2α, respectively. In the latter case the lysates from cells cultured in 1% O₂ for 24–72 h were pooled for analysis. α-Tubulin or β-actin was used as a loading control. The lanes in the C-P4H-II and HIF-1α panel in C were regrouped from different parts of the same Western blot.