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. 2003 Jan;14(1):173–189. doi: 10.1091/mbc.E02-06-0354

Figure 6.

Figure 6

(A) Analysis of αvβ3 and CD44 expression in WT and OPN−/− osteoclasts. Osteoclasts isolated from OPN−/− (lanes 1, 3, 5, and 7) and WT (lanes 2, 4, 6, and 8) were surface-labeled with biotin and immunoprecipitated with either anti-β3 (lanes 1 and 2) or anti-CD44 (lanes 5–8) antibodies. Immunoprecipitations with nonimmune serum (NI) are shown in lanes 3 and 4. Immunoprecipitates (lanes 1–6) were blotted with streptavidin-HRP to visualize the surface expression of the β3 subunit (lanes 1 and 2) and CD44 (lanes 5 and 6). The CD44 immunoblot (lanes 5 and 6) was stripped and immunoblotted with anti-CD44 (antibody to the sCD44; BioSource International Inc.) and anti–rat, HRP-conjugated primary and secondary antibodies, respectively. Only a single band of 85-kDa CD44 protein was detected in equal amounts in both OPN-deficient (lane 7) and WT (lane 8) osteoclast lysates. Arrows point to the CD44, αv, and β3 proteins. The results shown are representative of three different experiments. (B) The effect of OPN on CD44 surface expression. Cell lysates made from PBS (lanes 1 and 3) or OPN–treated (lanes 2 and 4) osteoclasts isolated from WT (lanes 1, 2, and 5) or OPN−/− (lanes 3 and 4) mice were immunoprecipitated with anti-CD44 (lanes 1–4) or nonimmune serum (NI; lane 5). Immunoprecipitates were blotted with streptavidin-HRP to visualize the surface expression of CD44 (lanes 1–5, top panel). The immunoblot shown in the top panel was stripped and blotted with an sCD44 antibody (BioSource International Inc.) to demonstrate the cellular levels of CD44 immunoprecipitated (bottom panel). The results shown are representative of three independent experiments. (C) Analysis of CD44 variant expression in osteoclasts. Lysates were made from osteoclasts (lanes 2, 3, 5, and 6) and melanoma cells, a positive control for CD44 variants (lanes 1 and 4). Immunoprecipitates were prepared using antibodies to CD44s (lanes 1–3; antibody to the sCD44; BioSource International Inc.) or CD44 v3–10 (lanes 4–6; goat polyclonal anti-human CD44 variant, v3–10; BioSource International Inc). Immunoprecipitates were blotted with streptavidin-HRP to visualize the surface expression of CD44. The arrow indicates the band corresponding to the size of sCD44. Asterisks indicate the splice variant form of CD44. The results shown are representative of three independent osteoclast preparations and experiments.