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Figure 8


Fig. 8. Overexpression of cortactin mutants decreases cortactin phosphorylation in podosomes. RAW-derived osteoclasts were co-transfected with GFP-actin and corWT-flag or GFP-actin and cor3YF-flag. Cells were fixed and stained for the flag tag. (A) CorWT and (B) cor3YF localized to podosomes. (C) Cells were fixed and stained for cortactin and cortactin pY421. Five control cells, eight cells expressing corWT and eight cells expressing cor3YF were segmented and the fluorescence intensity of their podosomes was determined. In control cells, the average cortactin intensity calculated (±s.d.) was 61±27 (cortactin) and 432±132 (cortactin pY421). In cells overexpressing corWT, the average intensities calculated were 638±288 (cortactin) and 811±271 (cortactin pY421). In cells overexpressing cor3YF, the average cortactin intensity calculated (±s.d.) was 512±230 (cortactin) and 501±189 (cortactin pY421). Note the ten- or eightfold increase in cortactin intensity in cells expressing corWT or cor3YF, respectively, and only a 45% or 16% increase in cortactin phosphorylation in these cells. (D) The ratio of phosphocortactin to general cortactin intensities.





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