Figure 2.

pSer⁷⁸⁴-VCP Is the DNA-Damage-Induced Nuclear Antigen of the pSer¹³⁷-Pfn1 Antibody

(A) HeLa cells were treated with DMSO, 200 nM of SN38, and 5 μM of etoposide for 6 h, followed by staining using the pSer¹³⁷-Pfn1 antibody. DAPI was used to stain DNA.

(B) HeLa cells were treated with DMSO, 200 nM of SN38, and 1 μM of gemcitabine for 16 h, followed by western blot analysis using the pSer¹³⁷-Pfn1 antibody. Red arrowhead indicates the DNA-damage-induced ~100-kDa protein.

(C) Nuclear extracts of DMSO- or SN38-treated (200 nM, 16 h) HeLa cells were immunoprecipitated by the pSer¹³⁷-Pfn1 antibody, followed by SDS-PAGE analysis and silver staining. The drug-induced protein at ~100 kDa was excised and identified as VCP by mass spectrometry (Held et al., 2013).

(D) Pull-down samples from (C) were analyzed by western blot using a VCP-specific antibody.

(E) HeLa cells were infected with shLuc or two distinct shVCPs for 3 days, treated with 5 μM of etoposide for 6 h, and immunostained using the pSer¹³⁷-Pfn1 antibody.

(F) HeLa cells were treated with 5 μM of etoposide for 6 h, extracted by the CSK buffer for 5 min, and stained by the pSer¹³⁷-Pfn1 antibody.

(G) HEK293T cells were transfected with wild-type or mutant VCP-GFP, treated with 200 nM of SN38 for 16hr, and immunoprecipitated using the pSer¹³⁷-Pfn1 antibody. Pull-down and input samples were analyzed by western blot using a GFP antibody.

(H) Input samples from (A) were analyzed by western blot using the pSer¹³⁷-Pfn1 or VCP antibodies.

(I) HeLa cells were pre-treated for 30 min with DMSO, 10 mM of caffeine, or 10 μM of KU-55933, followed by 30 min of treatment with 50 μM of etoposide. They were lysed by RIPA (without SDS), and the soluble and insoluble fractions were analyzed by western blot. pSer⁷⁸⁴-VCP was detected by the pSer¹³⁷-Pfn1 antibody.

All data, except (C), were independently confirmed two to three times. Scale bars, 20 μm (A) and (E) and 10 μm (F). See also Figures S4 and S5.