Figure 1.

(A) TRAP stain of wild-type (WT) and Mitf^mi/mi mouse osteoclasts. Wild-type mouse osteoclasts were grown from spleen in the presence of serum, M-CSF, and 150 ng/ml RANKL (panel 1) or without RANKL (panel 2). At day 9 of culture the cells were stained for TRAP. TRAP-positive cells failed to form in the absence of RANKL. Day 9 wild-type (panel 3) and Mitf^mi/mi (panel 4) mouse osteoclasts grown in complete medium (containing RANKL) and stained for TRAP. TRAP-positive osteoclasts were successfully generated from Mitf^mi/mi splenocytes, although TRAP intensity and number of nuclei per cell were somewhat diminished. (B) Mitf^mi/mi mutant osteoclasts contain decreased levels of cathepsin K protein. Total protein from day 10 osteoclast cultures of wild-type or Mitf^mi/mi osteoclasts was subjected to Western blotting with anti-cathepsin K (2-min and a longer 10-min Western blot exposures are shown) and antitubulin antibodies. (C) Mitf^mi/mi mutant osteoclasts contain decreased levels of cathepsin K mRNA. Poly(A)⁺ RNA from day 10 osteoclast cultures of wild-type or Mitf^mi/mi osteoclasts was analyzed by Northern blotting using probes for mouse cathepsin K, c-fms, RANK, and GAPDH.