Fig. 6. cSIRP expressed in neurons is phosphorylated and binds SHP-2. Cortical neurons transfected with GFP-cSIRP were cultured for 48 hours. Cells and neurites were harvested, permeabilized with ß-escin and incubated for 10 minutes at 37°C in the presence or absence of ATP and vanadate. GFP-cSIRP was immunoprecipitated from a TX100 extract of the samples and analyzed by western blot (IB; =anti). Probing with anti-pTyr antibody (Cy5-conjugated secondary antibody) revealed greatly increased phosphorylation of a single band in the ATP plus vanadate-treated samples (top panel). The blot was stripped, checked for absence of remaining Cy5 signal, and re-probed with anti-GFP (middle panel). This confirmed (i) presence of approximately equal amounts of GFP-cSIRP in both samples, and (ii) the identity of the pTyr-positive band. The same blot was also probed with anti-SHP-2 (Cy3-conjugated secondary antibody), revealing SHP-2 co-precipitation in ATP plus vanadate samples only. Representative image of two independent experiments are shown.