Figure 1.
Figure 1. VEGFR2 stability is controlled by SCFβ-TRCP. (A) Immunoblot analysis of WCLs and immunoprecipitates (IP) derived from 293T cells transfected with HA-VEGFR2 and various Myc-tagged Cullin constructs or EV. Data shown is representative of two independent experiments. (B) Immunoblot analysis of HMVEC WCLs and anti-VEGFR2 immunoprecipitates. Mouse IgG was used as a negative control for the immunoprecipitation procedure. Data shown is representative of two independent experiments. (C) Immunoblot analysis of HMVECs infected with the shRNA constructs specific for GFP or the indicated Cullin constructs. Data shown is representative of two independent experiments. (D) Immunoblot analysis of immunoprecipitates and WCL derived from 293T cells transfected with HA-VEGFR2 and/or Myc-tagged β-TRCP1, β-TRCP2, or β-TRCP1 (R474A) constructs. Data shown is representative of two independent experiments. (E) Immunoblot analysis of HMVEC WCLs and anti-VEGFR2 immunoprecipitates. Mouse IgG was used as a negative control for the immunoprecipitation procedure. Data shown is representative of two independent experiments. (F) Immunoblot analysis of WCL and immunoprecipitates derived from 293T cells transfected with HA-VEGFR2 and Myc–β-TRCP1 constructs as indicated. Where indicated, cell lysates were pretreated with λ-phosphatase before the immunoprecipitation procedure. Data shown is representative of two independent experiments. (G) Immunoblot analysis of WCL from 293T and HeLa cells transfected with shRNA constructs specific for GFP or β-TRCP1, β-TRCP2, or β-TRCP1+2. Data shown is representative of two independent experiments. (H) 293T cells were transfected with the indicated shRNA constructs. 20 h after transfection, cells were split into 60-mm dishes. 20 h later, 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 three independent experiments. (I) Quantification of the band intensities in H. VEGFR2 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n = 3).

VEGFR2 stability is controlled by SCFβ-TRCP. (A) Immunoblot analysis of WCLs and immunoprecipitates (IP) derived from 293T cells transfected with HA-VEGFR2 and various Myc-tagged Cullin constructs or EV. Data shown is representative of two independent experiments. (B) Immunoblot analysis of HMVEC WCLs and anti-VEGFR2 immunoprecipitates. Mouse IgG was used as a negative control for the immunoprecipitation procedure. Data shown is representative of two independent experiments. (C) Immunoblot analysis of HMVECs infected with the shRNA constructs specific for GFP or the indicated Cullin constructs. Data shown is representative of two independent experiments. (D) Immunoblot analysis of immunoprecipitates and WCL derived from 293T cells transfected with HA-VEGFR2 and/or Myc-tagged β-TRCP1, β-TRCP2, or β-TRCP1 (R474A) constructs. Data shown is representative of two independent experiments. (E) Immunoblot analysis of HMVEC WCLs and anti-VEGFR2 immunoprecipitates. Mouse IgG was used as a negative control for the immunoprecipitation procedure. Data shown is representative of two independent experiments. (F) Immunoblot analysis of WCL and immunoprecipitates derived from 293T cells transfected with HA-VEGFR2 and Myc–β-TRCP1 constructs as indicated. Where indicated, cell lysates were pretreated with λ-phosphatase before the immunoprecipitation procedure. Data shown is representative of two independent experiments. (G) Immunoblot analysis of WCL from 293T and HeLa cells transfected with shRNA constructs specific for GFP or β-TRCP1, β-TRCP2, or β-TRCP1+2. Data shown is representative of two independent experiments. (H) 293T cells were transfected with the indicated shRNA constructs. 20 h after transfection, cells were split into 60-mm dishes. 20 h later, 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 three independent experiments. (I) Quantification of the band intensities in H. VEGFR2 band intensity was normalized to tubulin, and then normalized to the t = 0 controls. The error bars represent mean ± SD (n = 3).

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