Figure 4
Figure 4. Lyn is associated with c-Cbl and regulates its stability in CML cells. (A) K562 and K562R cells were subjected to electroporation with control or Lyn-specific siRNA, and after 48 hours changes in phosphotyrosine levels were accessed in equal protein lysates by immunoblotting. Migration of molecular mass standards is shown on the left, and individual targets are shown on the right. In addition to a reduction in pY-Lyn levels in Lyn siRNA-treated cells, Lyn down-regulation in K562R cells was associated with differential regulation of 2 phosphoproteins of 75 kDa and 130 kDa. (B) Lyn and Hck were found to be highly expressed in a lymphoid blast crisis CML patient who failed imatinib therapy (Figure 2B). A specimen was obtained and treated with imatinib (5 μM) or dasatinib (0.5 μM) for 2 hours before lysates (1 mg) were prepared and subjected to Lyn (left) or Hck (right) immunoprecipitation. Immune complexes were resolved by SDS-PAGE and immunoblotted for phosphotyrosine. In addition to recovery of Lyn or Hck by immunoprecipitation, 2 phosphotyrosine protein bands (130 kDa, 75 kDa) were detected in control untreated cells. Imatinib-reduced phosphorylation or recovery of the 130-kDa protein in either Lyn or Hck immunoprecipitates, while dasatinib reduced phosphorylation or recovery of both the 130-kDa and 75-kDa phosphoproteins. In a parallel experiment, the Lyn coprecipitating 130-kDa band was excised from a silver-stained gel, subjected to trypsinization, and liquid chromatography/mass spectrometry (LC/MS) analysis. The 130-kDa protein was identified as c-Cbl. The 75-kDa protein has not yet been identified. (C) To confirm c-Cbl as a Lyn-associated and -regulated protein, K562R cells were subjected to Lyn silencing and (first panel) total lysates were immunoblotted for phosphotyrosine, (second panel) total lysates were immunoblotted for c-Cbl, (third panel) c-Cbl immunoprecipitates or total lysate (TL) was immunoblotted for phosphotyrosine, and (fourth panel) total lysates were subjected to c-Cbl immunoprecipitation and Lyn immunoblotting. The migration of specific target proteins is shown on the right. (D) Lyn was immunoprecipitated from protein lysates (250 mg) derived from 2 CML myeloid blast crisis patient specimens and immunoblotted for c-Cbl.

Lyn is associated with c-Cbl and regulates its stability in CML cells. (A) K562 and K562R cells were subjected to electroporation with control or Lyn-specific siRNA, and after 48 hours changes in phosphotyrosine levels were accessed in equal protein lysates by immunoblotting. Migration of molecular mass standards is shown on the left, and individual targets are shown on the right. In addition to a reduction in pY-Lyn levels in Lyn siRNA-treated cells, Lyn down-regulation in K562R cells was associated with differential regulation of 2 phosphoproteins of 75 kDa and 130 kDa. (B) Lyn and Hck were found to be highly expressed in a lymphoid blast crisis CML patient who failed imatinib therapy (Figure 2B). A specimen was obtained and treated with imatinib (5 μM) or dasatinib (0.5 μM) for 2 hours before lysates (1 mg) were prepared and subjected to Lyn (left) or Hck (right) immunoprecipitation. Immune complexes were resolved by SDS-PAGE and immunoblotted for phosphotyrosine. In addition to recovery of Lyn or Hck by immunoprecipitation, 2 phosphotyrosine protein bands (130 kDa, 75 kDa) were detected in control untreated cells. Imatinib-reduced phosphorylation or recovery of the 130-kDa protein in either Lyn or Hck immunoprecipitates, while dasatinib reduced phosphorylation or recovery of both the 130-kDa and 75-kDa phosphoproteins. In a parallel experiment, the Lyn coprecipitating 130-kDa band was excised from a silver-stained gel, subjected to trypsinization, and liquid chromatography/mass spectrometry (LC/MS) analysis. The 130-kDa protein was identified as c-Cbl. The 75-kDa protein has not yet been identified. (C) To confirm c-Cbl as a Lyn-associated and -regulated protein, K562R cells were subjected to Lyn silencing and (first panel) total lysates were immunoblotted for phosphotyrosine, (second panel) total lysates were immunoblotted for c-Cbl, (third panel) c-Cbl immunoprecipitates or total lysate (TL) was immunoblotted for phosphotyrosine, and (fourth panel) total lysates were subjected to c-Cbl immunoprecipitation and Lyn immunoblotting. The migration of specific target proteins is shown on the right. (D) Lyn was immunoprecipitated from protein lysates (250 mg) derived from 2 CML myeloid blast crisis patient specimens and immunoblotted for c-Cbl.

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