Figure 3.
Osteoblast phenotype in Src+/+ and Src−/− immortalized cells. (a) Src protein expression in immortalized osteoblasts. Confluent monolayers were lysed and 60 μg of total cell protein was electrophoresed in a 10% SDS-PAGE, blotted to nitrocellulose filter paper, and probed with Src pAb (diluted 1:250) at 4°C overnight, followed by the HRP-conjugated secondary antibody (diluted 1:5,000) at 37°C for 1 h. The filter was stripped and reprobed with anti-actin pAb (diluted 1:250). Bands were detected by ECL. This evaluation was repeated three times with similar results. (b) 90% confluent Src+/+ and Src−/− immortalized osteoblasts were cultured for the indicated times and subjected to determination of ALP, as described in the legend to Fig. 2. Cultures were treated with or without 10−7 M dexamethasone (Dex) for the entire time frame. Positive nodules are evident as dark spots. This experiment was repeated three times with similar results. (c) 90% confluent Src+/+ and Src−/− immortalized osteoblasts were cultured for 3 wk in the presence of ascorbic acid and β-glycerophosphate, as described in the Materials and Methods and with or without 10−7 M Dex for the entire time frame. Cultures were then fixed and subjected to von Kossa staining for the detection of nodule mineralization (dark areas). Quantitative analysis was performed by scanning densitometry, as described in the Materials and Methods, and data from three independent experiments were expressed as arbitrary densitometric units (mean ± SEM). ***P < 0.001 versus Src+/+; ND, non detectable.