Lyn is a Shp2 substrate in vitro and in vivo. (A) Lyn protein was immunoprecipitated from IL-3–starved Ba/F3GR cells washed twice with lysis buffer and once with phosphatase buffer, then mixed with 10 μg of purified GST, GST-Shp2, GST-Shp2C459S, or 5, 10, or 15 μg of GST-Shp2E76A in 200 μL of phosphatase buffer. Western blotting was performed with anti-phospho-Lyn Tyr507 antibody and reprobed with anti-Lyn antibody. The results showed that activated form of Shp2E76A directly dephosphorylated Tyr507 of Lyn, whereas wild-type Shp2 and constitutively inactivated Shp2 C459S had no or weak ability to dephosphorylate Tyr507 of Lyn. (B) Ba/F3GR cells were starved of IL-3, pretreated with increasing concentration of phosphatase inhibitor Na₃VO₄ for 1 hour, and followed by stimulation with 100 ng/mL G-CSF for 5 minutes. Cell lysates were blotted with anti-phospho-LynY507 and reprobed with anti-Lyn antibody. (C) After transfection of MEF cells with HA-tagged G-CSFR, surface expression of G-CSFR was detected by flow cytometry; shaded histogram represents the staining with PE-conjugated-isotype antibody, gray outline represents the staining with PE-conjugated anti–human G-CSFR antibody. (D) The expression of HA-tagged human G-CSFR from wild-type and Shp2^−/− MEF cell lysates was detected by Western blot. (E) G-CSFR signaling was reconstituted in wild-type and Shp2^−/− MEF cells with the expression of G-CSFR by detection of the phosphorylation of Stat3. (F) Dephosphorylation of phospho-Lyn Tyr507 occurred in G-CSFR–expressing MEF cells in response to G-CSF, whereas it was impaired in G-CSFR–expressing Shp2^−/− MEF cells. Numerical values are shown from densitometric analysis. (G) Shp2 (or empty vector as control) was stably transfected into Ba/F3GR cells, whole cell lysates were then prepared, and Western blot showed overexpression of Shp2 in Ba/F3GR cells. (H) Overexpression of Shp2 induced sustained dephosphorylation of phospho-Lyn Tyr507 and phosphorylation of Lyn Tyr396 in Ba/F3GR cells in response to G-CSF. (I) Proteins immunoprecipitated with Shp2 from G-CSF–treated Ba/F3GR cells were blotted for Lyn. Whole cell lysates (WCL) provided positive controls for Shp2 (top panel) and Lyn (bottom panel).