Figure 2.
Figure 2. CKI is involved in the regulation of VEGFR2 stability. (A) Immunoblot analysis of 293T cells transfected with HA-VEGFR2, Flag–β-TRCP1, and indicated kinases. Where indicated, cells were treated with the proteasome inhibitor MG132. Data shown is representative of two independent experiments. (B) Immunoblot analysis of 293T cells transfected with HA-VEGFR2 and/or Myc-CKIδ together with Flag-WT–β-TRCP1 or Flag-R474A–β-TRCP1. Data shown is representative of two independent experiments. (C and D) Immunoblot analysis of HMVECs treated with the CKI inhibitor D4476 at the indicated concentrations for 12 h. In D, where indicated, 100 ng/ml VEGF-A was added for 2 h before harvesting. Data shown is representative of two independent experiments. (E) Immunoblot analysis of WCLs and immunoprecipitates (IP) derived from 293T cells transfected with HA-VEGFR2 and Myc-tagged versions of indicated CKI isoforms. Data shown is representative of two independent experiments. (F) Immunoblot analysis of HMVECs infected with shRNA specific for GFP or the indicated CKI isoforms. Data shown is representative of two independent experiments. (G) HMVECs were transfected with the indicated shRNA constructs. 40 h after infection, cells were treated with 1 µg/ml puromycin for 72 h to eliminate the noninfected cells. Afterward, the resulting cells were split into 60-mm dishes and, after another 20 h, were treated with 20 µg/ml CHX. At the indicated time points, WCLs were prepared, and immunoblots were probed with the indicated antibodies. Data shown is representative of three independent experiments. (H) Quantification of the band intensities in G. VEGFR2 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n = 3). (I) 293T cells were transfected with EV or a construct encoding CKIδ. 20 h after transfection, cells were split into 60-mm dishes, and after another 20 h cells were treated with 20 µg/ml CHX. At the indicated time points, WCLs were prepared, and immunoblots were probed with the indicated antibodies. Data shown is representative of two independent experiments.

CKI is involved in the regulation of VEGFR2 stability. (A) Immunoblot analysis of 293T cells transfected with HA-VEGFR2, Flag–β-TRCP1, and indicated kinases. Where indicated, cells were treated with the proteasome inhibitor MG132. Data shown is representative of two independent experiments. (B) Immunoblot analysis of 293T cells transfected with HA-VEGFR2 and/or Myc-CKIδ together with Flag-WT–β-TRCP1 or Flag-R474A–β-TRCP1. Data shown is representative of two independent experiments. (C and D) Immunoblot analysis of HMVECs treated with the CKI inhibitor D4476 at the indicated concentrations for 12 h. In D, where indicated, 100 ng/ml VEGF-A was added for 2 h before harvesting. Data shown is representative of two independent experiments. (E) Immunoblot analysis of WCLs and immunoprecipitates (IP) derived from 293T cells transfected with HA-VEGFR2 and Myc-tagged versions of indicated CKI isoforms. Data shown is representative of two independent experiments. (F) Immunoblot analysis of HMVECs infected with shRNA specific for GFP or the indicated CKI isoforms. Data shown is representative of two independent experiments. (G) HMVECs were transfected with the indicated shRNA constructs. 40 h after infection, cells were treated with 1 µg/ml puromycin for 72 h to eliminate the noninfected cells. Afterward, the resulting cells were split into 60-mm dishes and, after another 20 h, were treated with 20 µg/ml CHX. At the indicated time points, WCLs were prepared, and immunoblots were probed with the indicated antibodies. Data shown is representative of three independent experiments. (H) Quantification of the band intensities in G. VEGFR2 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n = 3). (I) 293T cells were transfected with EV or a construct encoding CKIδ. 20 h after transfection, cells were split into 60-mm dishes, and after another 20 h cells were treated with 20 µg/ml CHX. At the indicated time points, WCLs were prepared, and immunoblots were probed with the indicated antibodies. Data shown is representative of two independent experiments.

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