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. Author manuscript; available in PMC: 2013 Mar 8.
Published in final edited form as: J Immunol. 2009 Jul 10;183(3):1804–1812. doi: 10.4049/jimmunol.0804206

Figure 5. The tyrosine and proline rich regions of SLP-76 mediate OC spreading and function.

Figure 5

(A) Retroviral SLP-76 constructs used: WT tyrosine to phenylalanine mutation (3YF), proline-rich region (PRR) deletion (Δ157-222), and SH2 point mutation (R448K). (B–G) DKO BMMs were transduced with the four SLP-76 constructs or empty vector. (B) Following selection, the cells were plated with M-CSF and RANKL on plastic. After 5 days, OCs were stained for TRAP activity. (C) The number of spread cells in (B) was quantified on days 4 and 5. (D) Actin ring formation by transduced cells, generated on bone, visualized by FITC-phalloidin staining. (E) The percentage of cells exhibiting actin rings on day 5 was quantified. (F) After removal of OCs generated on bone, resorptive lacunae were stained with peroxidase-conjugated WGA and 3-3′-diaminobenzidine and (G) Resorptive pit area was quantified histomorphometrically. (H) DKO BMMs were transduced with GFP alone (empty vector), GFP-WT-SLP-76 or GFP-tagged mutant constructs. Cells were cultured on glass coverslips with M-CSF and RANKL for five days to generate OCs. The cells were examined by fluorescent microscopy to determine GFP distribution. (*p<0.05, **p<0.01, ***p<0.001 vs WT) All images were acquired at 200x magnification.