American Society of Hematology

Figure 1.

$Figure 1. Altered expression, subcellular localization, tyrosine phosphorylation of Shp2 in human leukemia cell lines. (A) Cell lysates (20 μg total proteins) were analyzed by Western blot with antibodies against Shp2 or β-actin, as a control for equal sample loading. (B) For normal hematopoietic cell samples (PBMNCs), 30 μg total proteins were loaded for Western blot analysis for Shp2 and β-actin, while 5 μg total proteins from NB4 leukemia cell lysate was used as a positive control for the 2 Shp2 bands and for comparison of the relative Shp2 contents. (C) The histogram shows p-Shp2 protein levels normalized against the actin expression in leukemia cell lines and normal PBMNC samples. Data are representative of 3 independent experiments. (D) Leukemic cells were lysed in hypotonic buffer, and lysates were used to prepare total cellular protein (T), cytosolic (C), membrane-soluble (M), and nucleus (N) fractions as described in the text. An aliquot of each fraction (20 μg total proteins) was subjected to immunoblot analysis for Shp2. (E) Proteins were immunoprecipitated from each fraction with an anti-Shp2 antibody and subjected to immunoblot analysis with an antiphosphotyrosine (pTyr) antibody. IP indicates immunoprecipitation. Data are representative of 2 independent experiments.$

Altered expression, subcellular localization, tyrosine phosphorylation of Shp2 in human leukemia cell lines. (A) Cell lysates (20 μg total proteins) were analyzed by Western blot with antibodies against Shp2 or β-actin, as a control for equal sample loading. (B) For normal hematopoietic cell samples (PBMNCs), 30 μg total proteins were loaded for Western blot analysis for Shp2 and β-actin, while 5 μg total proteins from NB4 leukemia cell lysate was used as a positive control for the 2 Shp2 bands and for comparison of the relative Shp2 contents. (C) The histogram shows p-Shp2 protein levels normalized against the actin expression in leukemia cell lines and normal PBMNC samples. Data are representative of 3 independent experiments. (D) Leukemic cells were lysed in hypotonic buffer, and lysates were used to prepare total cellular protein (T), cytosolic (C), membrane-soluble (M), and nucleus (N) fractions as described in the text. An aliquot of each fraction (20 μg total proteins) was subjected to immunoblot analysis for Shp2. (E) Proteins were immunoprecipitated from each fraction with an anti-Shp2 antibody and subjected to immunoblot analysis with an antiphosphotyrosine (pTyr) antibody. IP indicates immunoprecipitation. Data are representative of 2 independent experiments.

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