Characterization of SHP-1 expression and localization in ES cells. (A) Whole cell lysate (WCL) was prepared from 5 × 106 cells (B = Ba/F3, E = E14tg2a ES cells, O = OP-9), and SHP-1 immunoprecipitates (α-SHP-1 IP) were prepared. These immunoprecipitates were separated by SDS-PAGE and immunoblotted for SHP-1. Blots were stripped and reprobed for SHP-2. Arrows indicate the positions of SHP-1 and SHP-2. (B) ES cells (1 × 104) were cytospun onto a slide, and immunocytochemistry was performed using anti–SHP-1 antibody. As a control, a duplicate slide was prepared and probed with α-SHP-1 + blocking peptide (α-SHP-1 + B.P.) original magnification ×400, a 40×/10.65 objective lens was used. (C) Total cell extracts (T), NP40 soluble extracts comprising cytosolic and membrane proteins (C/M), and nuclear fractions (N) were generated from 1 × 107 ES cells, and anti–SHP-1 immunoprecipitate (IP) was prepared. Extracts and precipitates were separated by SDS-PAGE and probed with the antiphosphotyrosine antibody 4G10 (α-pY, top blot). The equivalent of 1 × 105 cells was loaded per lane for total extract, and the equivalent of 2 × 105 cells was loaded per lane for the cytosolic and nuclear fractions (ie, double the amount of total extract separated). The blot was stripped and reprobed with an anti–SHP-1 antibody (bottom blot). (D) Extracts from each subcellular fraction (loaded in a manner equivalent to that in panel C) were separated on duplicate gels, 7.5% acrylamide for anti-gp130 immunoblotting and 10% acrylamide for anti–Oct-4. These blots were stripped and reprobed with antibodies against STAT3 and SHP-2, respectively. The positions of tyrosine-phosphorylated proteins coprecipitating with SHP-1 are indicated. Molecular weight standards are indicated on the left side of the panels. IgGH indicates immunoglobin heavy chain.
