American Society of Hematology

Fig. 6.

$Fig. 6. Cdk2-and cdk4-associated kinase activities, and expression and phosphorylation status of the retinoblastoma gene product (pRb) in M1 and M1E2F-1 cells, untreated and treated with IL-6. / (A) Cdk2- and cdk4-associated kinase activities in M1 and M1E2F-1 after treatment with IL-6. Cell lysates were prepared at the indicated times after addition of IL-6 and kinase reactions were carried out as described in “Materials and methods.” One hundred micrograms protein per sample and histone H1 substrate were used in the cdk2 kinase assay. For the cdk4 kinase assays, 400 μg protein per sample and GST-Rb substrate were used. After completion of the reactions, samples were fractionated on a 10% gel, dried, and subjected to autoradiography for 2 hours. (B) Western blot analysis of the expression and phosphorylation status of the pRb in IL-6–treated M1 and M1E2F-1 cells. Protein extracts were prepared before and after induction for differentiation with IL-6 at the indicated time points as described in “Materials and methods.” Fifty micrograms per lane of total protein extract were fractionated on a 6% SDS-PAGE gel, transferred to a PVDF membrane, and probed with an anti-Rb monoclonal antibody (diluted 1:1000). Signals were developed by using the enhanced chemiluminescence (ECL) Western blotting system (Amersham). ppRb indicates the hyperphosphorylated state and Rb is the hypophosphorylated form.$

Cdk2-and cdk4-associated kinase activities, and expression and phosphorylation status of the retinoblastoma gene product (pRb) in M1 and M1E2F-1 cells, untreated and treated with IL-6.

(A) Cdk2- and cdk4-associated kinase activities in M1 and M1E2F-1 after treatment with IL-6. Cell lysates were prepared at the indicated times after addition of IL-6 and kinase reactions were carried out as described in “Materials and methods.” One hundred micrograms protein per sample and histone H1 substrate were used in the cdk2 kinase assay. For the cdk4 kinase assays, 400 μg protein per sample and GST-Rb substrate were used. After completion of the reactions, samples were fractionated on a 10% gel, dried, and subjected to autoradiography for 2 hours. (B) Western blot analysis of the expression and phosphorylation status of the pRb in IL-6–treated M1 and M1E2F-1 cells. Protein extracts were prepared before and after induction for differentiation with IL-6 at the indicated time points as described in “Materials and methods.” Fifty micrograms per lane of total protein extract were fractionated on a 6% SDS-PAGE gel, transferred to a PVDF membrane, and probed with an anti-Rb monoclonal antibody (diluted 1:1000). Signals were developed by using the enhanced chemiluminescence (ECL) Western blotting system (Amersham). ppRb indicates the hyperphosphorylated state and Rb is the hypophosphorylated form.

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