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. 2016 Jan 8;7(5):6088–6104. doi: 10.18632/oncotarget.6842

Figure 5. Cdk5 regulates NICD generation.

Figure 5

(A) Immunoblots from HUVECs transfected with nt (non-targeting) and Cdk5 siRNA and plated onto PBS- or DLL4-coated dishes probed with anti-NICD, anti-Cdk5, and anti-actin antibodies are shown. n = 3. The bar graph displays the quantification of the NICD immunoblot normed to the loading control (One Way ANOVA, Tukey, SEM, *p ≤ 0.05). (B) Immunoblots from HUVECs untreated (co) or treated with roscovitine (rosco, 10 μM), plated onto PBS- or Dll4-coated dishes and probed with anti-NICD and anti-actin antibodies are shown. n = 3. The bar graph displays the quantification of the NICD immunoblot normed to the loading control (One Way ANOVA, Tukey, SEM, *p ≤ 0.05). (C) Expression of Notch1 receptor mRNA of HUVECs transfected with nt (non-targeting) or Cdk5 siRNA is shown. One-Way ANOVA, Tukey, n = 3. (D) Immunoblots from HUVECs transfected with nt (non-targeting) or Cdk5 siRNA and probed with anti-Notch, anti-Cdk5, and anti-tubulin antibodies are shown. n = 3. (E) Immunoblots of HUVECs silenced with nt (non-targeting) or Cdk5 siRNA and treated with MG132 for 24 h before plating onto Dll4 and probed with anti-NICD, anti-Cdk5 and anti-actin antibodies are shown. n = 3. The bar graph displays the quantification of the NICD immunoblot normed to the loading control (One Way ANOVA, Tukey, SEM, *p ≤ 0.05). (F) Immunoblots of HUVECs treated with MG132 for the indicated timepoints and probed with anti-β-catenin and anti-actin antibodies are shown. n = 2. The bar graph displays the quantification of the NICD immunoblot normed to the loading control (One Way ANOVA, Tukey, SEM, ns: not significant). (G) Immunoblots of HUVECs overexpressing NICD or empty vector treated with/without roscovitine for the indicated times and probed with anti-NICD and anti-tubulin antibodies are shown. n = 3. (H) Immunoblots of HUVECs transfected with nt (non-targeting) or Cdk5 siRNA plated onto Dll4 and probed with anti-presenilin, anti-Cdk5, and anti-actin antibodies are shown. Phosphorylated and total presenilin is denoted by the upper and lower band and marked by arrows. n = 3. (I) Immunoblots of HUVECs treated with/without roscovitine at indicated concentrations plated onto Dll4 and probed with anti-presenilin and anti-actin antibodies are shown. Phosphorylated and total presenilin is denoted by the upper and lower band and marked by arrows. n = 3.