Figure 3.
Monoclonal pSer784-VCP Antibody Confirms the Nuclear Antigen of the pSer137-Pfn1 Antibody
(A) HeLa cells treated with DMSO or SN38 (200 nM, 16 h) were analyzed by western blot using the pSer137-Pfn1 or pSer784-VCP antibodies.
(B) HeLa cells treated with etoposide (50 μM, 1 h) were lysed, incubated at 37°C for 1 h with or without calf intestinal alkaline phosphatase, and analyzed by western blot using the pSer137-Pfn1, pSer784-VCP, or pan-VCP antibodies.
(C) HeLa cells treated with DMSO or etoposide (50 μM, 6 h) were subjected to immunoprecipitation by control immunoglobulin G (IgG) or pan-VCP antibody, followed by western blot using the pSer137-Pfn1, pSer784-VCP, and pan-VCP antibodies.
(D) HeLa cells treated with DMSO or SN38, as in (A), were subjected to immunoprecipitation using the pSer137-Pfn1 or pSer784-VCP antibodies, followed by western blot using a pan-VCP antibody.
(E) HeLa cells were treated with 200 nM of SN38 for 16 h and immunostained with the pSer784-VCP antibody.
(F) HeLa cells were treated with 50 μM of etoposide for 1 h, followed by double immunostaining using the pSer137-Pfn1 and pSer784-VCP antibodies.
(G and H) U2OS cells were treated with 50 μM of etoposide for 1 h, recovered for 90 min, and detergent-extracted before fixation and double staining by the pSer137-Pfn1 and 53BP1 antibodies (G) or pSer784-VCP and γH2AX (H) antibodies.
(I) Representative images in the SPECS TMA immunostained in parallel by the pSer137-Pfn1 and pSer784-VCP antibodies.
(J) Univariate Kaplan-Meier analysis of the SPECS TMA stained in (G). Nuclear Allred scores were binarized into low (0–4) versus high (5–8) groups. Two extra interpretable cases stained by the pSer137-Pfn1 antibody (n = 46) (but not by pSer784-VCP, n = 44) were included in the analysis. p values were based on Log-rank test.
More than 100 cells per experiment were analyzed for (D)–(F). Data in (A)–(H) have been independently confirmed 2-3 times. Scale bars, 20 μm (E), 4 μm (F–H), and 10 μm (I). See also Figure S5.