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. 2006 Jun;26(12):4539–4552. doi: 10.1128/MCB.02120-05

FIG. 2.

FIG. 2.

Primary osteoblasts are negative for E11 expression but begin to express this antigen with time in culture. MLO-Y4 osteocyte-like cells express the highest levels of E11. E11 in primary periosteal osteoblasts is not detectable upon isolation but increases with time in culture (A). Cells were isolated from long bones of 1-week-old mice and either lysed after isolation or cultured for 3 to 7 days before processing for Western blot analysis. The left part shows the reaction with the 8.1.1 antibody, and the right part shows Ponceau S staining. Note the lack of expression in the freshly isolated cells but the increased expression with time in culture. Immunocytochemical staining for E11 in freshly isolated cytocentrifuged mouse osteoblasts (B) and osteoblasts cultured for 3 days and then subjected to collagenase treatment and cytocentrifuged (C) was performed. This experiment was performed to show that collagenase treatment of the freshly isolated cells or cultured cells was not removing or having an effect on E11 expression on the cell surface. The insert in the upper right part shows the negative control. Scale bar = 200 μm. E11 expression is much higher in the MLO-Y4 osteocyte-like cell line than in other cell lines, as determined by Western blotting (D). The osteocyte-like cell line MLO-Y4 (Y4), osteoblast-like cell lines MC3T3 (MC) and 2T3, late osteoblast-early osteocyte MLO-A5 (A5) cells, primary osteoblasts (OB), primary fibroblasts (FB), and the fibroblast-like cell line NIH 3T3 (NIH) were cultured for 3 to 4 days before lysis. The upper and middle parts show reaction with the 8.1.1 antibody. The film was exposed for a short period of time to visualize relative expression (top) and for a longer period of time to visualize any additional bands (middle). The lower part shows Ponceau S staining. Note that the highest expression was in the MLO-Y4 osteocyte-like cells compared to less or no expression in the other cell lines. Bands of various sizes were observed, suggesting different extents or forms of posttranslational modification. A 100-kDa band can be seen with the MLO-Y4 cell, MLO-A5 cell, and primary osteoblast lysates after longer exposure.