FIG. 9.
Capn4 deficiency correlates with p27Kip1 and cyclin D2 protein accumulation in proliferative chondrocytes in embryonic growth plates. Immunohistochemistry using anti-p27Kip1 and cyclin D2 antibodies showed stronger immunoreactivities of p27Kip1 (A and B) and cyclin D2 (C and D) in nuclei of mutant proliferating chondrocytes than in controls in vivo. (A) Experiments 1 to 3 represent three independent experiments using control and mutant littermate samples, respectively. Experiment 3 shows immunohistochemical analysis of periarticular chondrocytes by using Alexa Fluor-labeled goat anti-rabbit IgG antibody (Invitrogen) as a secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratory). (B and D) Semiquantitative representation of immunohistochemical analysis (n = 4 each group). Magnification is ×200. *, P < 0.05; **, P < 0.005; and ***, P < 0.001.