Figure 12.

MKP-1 and PKCζ colocalize in the nuclear compartment of vascular fibroblasts. (A) Representative photographs of MKP-1 and PKCζ in normoxic fibroblasts. Quiescent cells were fixed with cold 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Double immunofluorescent staining was performed with anti-MKP-1 and PKCζ antibodies. Magnification, ×100. (B) Representative photographs of nuclear localization of MKP-1 and PKCζ in hypoxic fibroblasts. Growth-arrested cells were exposed to hypoxia for 24 h. At the end of hypoxic exposure, MKP-1 and PKCζ were visualized by double immunofluorescent staining. (C) Nuclear staining of PKCζ is abolished in the presence of PKCζ peptide and anti-PKCζ antibody. To confirm the specificity of nuclear PKCζ staining, the PKCζ peptide was preincubated with the anti-PKCζ antibody overnight at 4°C. Immunofluorescent staining of PKCζ was performed with this antigen-antibody complex. (D) Myristoylated PKCζ pseudosubstrate peptide inhibitor induces disorganization of defined nuclear localization of MKP-1 in fibroblasts. Growth-arrested fibroblasts were preincubated with PKCζ-specific peptide inhibitor (10 μM) for 1 h at 37°C and then exposed to either normoxia or hypoxia for 24 h. At the end of the treatment, cells were fixed and processed for indirect immunofluorescent staining of MKP-1. Similar results were reproduced in two other fibroblast populations. Magnification, ×100. n = 4 replicate wells. A representative micrograph of the three independent experiments is shown in each case.