Phosphorylation of sDab1 and sDab1-Li by sFyn-B in transfected Cos1 cells in vitro. A: sDab1-GFP or sDab1-Li-GFP was transfected to Cos1 cells along with sFyn-B-GFP. 24 hours after transfection, cells were lysed and analyzed by western blotting using 4G10, anti-Dab1, anti-Fyn and anti-Tubulin. B: Lanes 1-4: sDab1-GFP, sDab1-Li-GFP, mDab1-wt (murine wild-type Dab1 as a positive control) or mDab1-5F (murine mutant form of Dab1 carrying phenylalanine substitutions at residues Y185F, Y198F, Y200F, Y220F and Y232F as a negative control) were expressed in Cos1 cells along with porcine wild-type Fyn-B. Lanes 5-8: PP2, a specific inhibitor of SFKs, was added to the same set of co-transfections as in lanes 1-4. Lanes 9-12: sDab1, sDab1-Li, mDab1-wt or mDab1-5F was separately transfected to Cos1 cells without any exogenous Fyn. 24 hours after transfection, cells were lysed and analyzed by Western blotting using anti Dab1-pY185, anti Dab1-pY198/200 and anti Dab1-pY232. The membranes were also probed with anti-Dab1, anti-Fyn and anti-Tubulin antibodies to confirm protein loading. At least three blots were analyzed for these experiments.
Long et al. BMC Neuroscience 2011 12:17 doi:10.1186/1471-2202-12-17