Fig. 6. ES cells express the s-SHIP... | American Society of Hematology

Fig. 6.

$Fig. 6. ES cells express the s-SHIP protein isoform that associates with the Grb2 adapter protein. / (A) Immunoprecipitation and immunoblot detection of s-SHIP in ES cell lysates. Lysate from ES-TL1 cells cultured in LIF was immunoprecipitated with the P2C6 anti–SHIP monoclonal antibody, separated on gels, transferred to membranes, and probed with P2C6, revealing 104-kd and 97-kd proteins (lane 5). No tyrosine phosphorylation of these proteins was detected when they were probed with the 4G10 anti–phosphotyrosine antibody. For comparison, lysates from 293T cells transfected with SHIP cDNA (lane 1) and s-SHIP cDNA (lane 2) were included in the blots. To further assess the tyrosine phosphorylation status of s-SHIP, timed LIF stimulation studies were performed. ES-TL1 cells incubated for 5 hours without LIF were stimulated with 2000 U/mL LIF for 0, 2, 5, or 10 minutes and were rapidly lysed. Equal amounts of total protein were immunoprecipitated with the P2C6 antibody and probed separately with the P2C6 and 4G10 antibodies (lanes 6-9). No tyrosine phosphorylation of s-SHIP was detected at any time point with the 4G10 antibody. For comparison, A20 B-lymphoid cells were stimulated with anti–IgG antibody for 0 or 5 minutes, lysed, immunoprecipitated with P2C6, and probed with P2C6 and 4G10 (lanes 3, 4), showing prominent tyrosine phosphorylation of SHIP at 5 minutes. Molecular mass standards are indicated on the right. (B) s-SHIP associates with Grb2 but not Shc in ES cells. ES-TL1 cell lysates were prepared and immunoprecipitated with the P2C6 anti–SHIP monoclonal antibody. Resolved immunoprecipitates were then blotted with antibodies specific for SHIP, Grb2, mSos1, or Shc (lane 1). Whole cell lysates from ES-TL1 cells were also included to confirm the expression of these proteins in ES cells (lane 2). (C) Grb2 associates with s-SHIP in ES cells. ES-TL1 cell lysate was prepared and immunoprecipitated with an anti–Grb2 polyclonal antibody. Resolved immunoprecipitate was then blotted with the P2C6 anti–SHIP monoclonal antibody (lane 3). For comparison, whole cell lysates from WEHI-231 cells (lane 1) and 293T cells transfected with s-SHIP cDNA (lane 2) were included. (D) Subcellular localization of s-SHIP protein in ES cells. Whole cell lysate, cytosol, and membrane fractions from ES-TL1 cells were prepared as described. One milligram total protein from each fraction was immunoprecipitated with the P2C6 anti–SHIP monoclonal antibody, and equal volumes of immunoprecipitate from each preparation were separated on gels, transferred, and blotted with the P2C6 anti–SHIP monoclonal antibody (lanes 2-4). For comparison, whole cell lysate from 293T cells transfected with s-SHIP cDNA was included (lane 1).$

ES cells express the s-SHIP protein isoform that associates with the Grb2 adapter protein.

(A) Immunoprecipitation and immunoblot detection of s-SHIP in ES cell lysates. Lysate from ES-TL1 cells cultured in LIF was immunoprecipitated with the P2C6 anti–SHIP monoclonal antibody, separated on gels, transferred to membranes, and probed with P2C6, revealing 104-kd and 97-kd proteins (lane 5). No tyrosine phosphorylation of these proteins was detected when they were probed with the 4G10 anti–phosphotyrosine antibody. For comparison, lysates from 293T cells transfected with SHIP cDNA (lane 1) and s-SHIP cDNA (lane 2) were included in the blots. To further assess the tyrosine phosphorylation status of s-SHIP, timed LIF stimulation studies were performed. ES-TL1 cells incubated for 5 hours without LIF were stimulated with 2000 U/mL LIF for 0, 2, 5, or 10 minutes and were rapidly lysed. Equal amounts of total protein were immunoprecipitated with the P2C6 antibody and probed separately with the P2C6 and 4G10 antibodies (lanes 6-9). No tyrosine phosphorylation of s-SHIP was detected at any time point with the 4G10 antibody. For comparison, A20 B-lymphoid cells were stimulated with anti–IgG antibody for 0 or 5 minutes, lysed, immunoprecipitated with P2C6, and probed with P2C6 and 4G10 (lanes 3, 4), showing prominent tyrosine phosphorylation of SHIP at 5 minutes. Molecular mass standards are indicated on the right. (B) s-SHIP associates with Grb2 but not Shc in ES cells. ES-TL1 cell lysates were prepared and immunoprecipitated with the P2C6 anti–SHIP monoclonal antibody. Resolved immunoprecipitates were then blotted with antibodies specific for SHIP, Grb2, mSos1, or Shc (lane 1). Whole cell lysates from ES-TL1 cells were also included to confirm the expression of these proteins in ES cells (lane 2). (C) Grb2 associates with s-SHIP in ES cells. ES-TL1 cell lysate was prepared and immunoprecipitated with an anti–Grb2 polyclonal antibody. Resolved immunoprecipitate was then blotted with the P2C6 anti–SHIP monoclonal antibody (lane 3). For comparison, whole cell lysates from WEHI-231 cells (lane 1) and 293T cells transfected with s-SHIP cDNA (lane 2) were included. (D) Subcellular localization of s-SHIP protein in ES cells. Whole cell lysate, cytosol, and membrane fractions from ES-TL1 cells were prepared as described. One milligram total protein from each fraction was immunoprecipitated with the P2C6 anti–SHIP monoclonal antibody, and equal volumes of immunoprecipitate from each preparation were separated on gels, transferred, and blotted with the P2C6 anti–SHIP monoclonal antibody (lanes 2-4). For comparison, whole cell lysate from 293T cells transfected with s-SHIP cDNA was included (lane 1).

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