Figure 5.

Claspin mRNA is regulated independently of the cell cycle but Claspin is responsive to NF-κB activation. (A) U2OS cells were depleted of IKKα, IKKβ, Claspin or Cyclin D1 using siRNA. WCLs were subjected to western blot analysis for the levels of the indicated proteins. (B) Quantitative RT–PCR analysis of Claspin mRNA prepared from U2OS cells depleted of IKKα, IKKβ, Claspin or Cyclin D1 by siRNA. ANOVA t-tests were performed on the means, and the P-values were calculated. ^**P⩽0.01 and ^***P⩽0.001. (C) U2OS cells were synchronized at G1/S using a double-thymidine block. Thymidine was subsequently washed and cells were released for the indicated times. Cells were analysed by flow cytometry and percentage of cells in each stage of the cell cycle is indicated. WCLs and RNA were prepared from these cells and analysed by western blot and quantitative RT–PCR, respectively. Specific antibodies and primer sets are indicated. (D) U2OS cells were synchronized at prometaphase using nocodazole. Nocodazole was subsequently washed and cells were analysed by flow cytometry and percentage of cells in each stage of the cell cycle is indicated. WCLs and RNA were prepared and analysed by western blot and quantitative RT–PCR, respectively. Specific antibodies and primer sets are indicated. (E) Quantitative RT–PCR analysis of Claspin mRNA prepared from U2OS cells depleted of β-TrCP by siRNA. (F) U2OS cells were treated with PMA (100 ng/ml)/Ionomycin (0.5 μM) for 4 h. One hour prior to harvesting, cells were treated with UV (40 J/m²) as indicated. WCLs were prepared and Claspin levels were analysed by western blot.