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. 2006 Mar 20;203(3):573–582. doi: 10.1084/jem.20051520

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Copyright © 2006, The Rockefeller University Press

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Figure 6. — Inhibition of PKC after IL-7 withdrawal blocks p27^Kip1 induction through a Skp2 and T187 phosphorylation–independent pathway. (A) Protein levels of p27^Kip1 and phospho-p27^Kip1 were measured in D1 cells. Cells were deprived of IL-7 for various time points and cell lysates were resolved by SDS-PAGE. Immunoblotting was performed with antibodies against p27^Kip1, phospho-p27^Kip1 (T187 or S10), and β-actin. (B) The effect of PKC inhibitor on p27^Kip1 stability was determined. IL-7 was withdrawn from D1 cells for 12 h and 5 μM of the PKC inhibitor Gö6850 was added at the time of IL-7 withdrawal. Protein levels were measured by immunoblotting using antibodies against p27^Kip1, phospho-p27^Kip1 (T187), and β-actin. 0.1% of DMSO was included as a negative control. (C) p27^Kip1 stability affected by phosphorylation was analyzed in D1 cells overexpressing the FLAG-tagged p27^Kip1 WT, single mutant T187A, S10A, or T187D, respectively. Extrogenous protein levels were detected by immunoblotting using antibody against FLAG. The GFP expression level was also measured as transfection efficiency control. (D) Skp2 and Cks1 expressions were measured in D1 cells. Cell lysates were prepared from cells deprived of IL-7 for 4 and 12 h, or cells cultured without IL-7 for 12 h and 5 μM of the PKC inhibitor Gö6850. Immunoblotting was analyzed by specific antibodies against Skp2, Cks1, and β-actin. 0.1% of DMSO was included as a negative control.