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

Figure 8.

Figure 8.

(A–E) Cell extracts from DMSO or melphalan-treated C33A2 cells were subjected to immunoprecipitation with the antibodies to phosphorylated BRCA1 (p-BRCA1) (A), hnRNP C (B) (C), Fip1 (D) or CPSF30 (E) followed by western blotting with antibodies to hnRNP C, CstF64 or Fip1 as indicated in the figure. The hours of DMSO or melphalan incubation of C33A2 cells are indicated on top of the gels. The levels of co-immunoprecipitated protein and the levels of each protein in the input extracts were quantified. Percent of input protein co-immunoprecipitated by each antibody in extracts from DMSO or melphalan treated cells are shown below each gel. For hnRNP C co-immunoprecipitations with anti-Fip1 or anti-CPSF30 antibody, the levels of co-immunoprecipiated hnRNP C in melphalan treated cells were divided by the levels of hnRNP C co-immunoprecipitated with anti Fip1 antibody or anti-CPSF30 in DMSO treated cells (D and E). (F) ChIP assays on DNA from C33A2 cells using antibody to hnRNP C and qPCR of the indicated HPV16 amplicons. 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. (G) 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 antibody to hnRNP C1 and the RNA extracted from the immunoprecipitated complexes was subjected to RT-PCR with primers that detect HPV16 E4 mRNAs spliced from SD880 to SA3358. (H) Western blot on extracts from C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to hnRNP C. hnRNP C levels were quantified in extracts prepared from cells transfected with the indicated siRNAs and divided by hnRNP C levels in extracts from cells transfected with scrambled siRNAs (scr). (I) sLuc activity produced by C33A2 cells transfected with scrambled siRNAs (scr) or siRNAs to hnRNP C followed by addition of 100 µM melphalan.