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. 2018 Mar 27;46(10):5029–5049. doi: 10.1093/nar/gky227

Figure 6.

Figure 6.

(A) Western blot on TRAP150 in C33A2 cells treated with DMSO (D) or with 100 µM melphalan (M) for the indicated time periods. TRAP150 levels were normalized to actin and TRAP150 over actin in untreated cells was set as 1. (B and C) Cell extracts from DMSO or melphalan treated C33A2 cells were subjected to immunoprecipitation with the antibodies to phosphorylated BRCA1 (p-BRCA1) (B) or TRAP150 (C), followed by western blotting with antibodies to TRAP150 or U2AF65, respectively. The hours of DMSO or melphalan incubation of C33A2 cells are indicated on top of the gels. The levels of immunoprecipitated protein and the levels of each protein in the input extracts were quantified. Percent of input protein immunoprecipitated by each antibody in extracts from DMSO or melphalan-treated cells are shown below each gel. (D) ChIP analyses on C33A2 cells using antibody to TRAP150 and qPCR of the indicated HPV16 amplicons. Primers and antibodies are listed in Supplementary Tables S3 and 4, respectively, and the location in the HPV16 genome of the PCR primers is shown in Supplementary Figure S5. Mean values with standard deviations of the amount of immunoprecipitated DNA compared to DNA from DMSO-treated cells are displayed. The qPCR values obtained for each primer pair with DNA extracted from DMSO-treated C33A2 cells were set to 1 to correct for differences between different ChIP extracts. Chip extracts were prepared from C33A2 cells treated with melphalan for the indicated time-periods. (E) C33A2 cells treated with DMSO or melphalan for the indicated time points were UV irradiated and subjected to CLIP assay as detailed in ‘Materials and Methods’ section. The RNA–protein complexes were immunoprecipitated with antibodies to U2AF65, BARD1, BCLAF1 or phosphorylated BRCA1 (p-BRCA1) and the RNA extracted from the immunoprecipitated complexes were subjected to RT-PCR with primers that detect HPV16 E4 mRNAs spliced from SD880 to SA3358. (F) Western blot on extracts from C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to BCLAF1, TRAP150 or U2AF65. Protein levels were quantified in extracts prepared from cells transfected with the indicated siRNAs and divided by protein levels in extracts from cells transfected with scrambled siRNAs (scr). (G) sLuc activity produced by C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to BCLAF1, TRAP150 or U2AF65 followed by addition of 100 µM melphalan.