Fig 1: Tpr maintains normal protein levels of GANP.a U-2 OS cells were transfected using siRNAs and either nontreated (Mock) or treated with 2 mM hydroxyurea (HU) for 4 h. RPA, loading control. Data are representative of three independent experiments. b U-2 OS cells were transfected using siRNAs and treated as in a. RPA, loading control. The experiment was performed twice yielding similar results. c U-2 OS cells were transfected using siRNAs. Tubulin, loading control. Data are representative of three independent experiments. d U-2 OS cells were transfected using siRNA targeting endogenous Tpr (siTPR #54), GANP (siGANP #87), or control siRNA (siCTRL). In parallel, cells were cotransfected with siRNA-resistant Tpr-GFP insensitive to siTPR #54. Tpr-GFP and GANP protein levels were analyzed by immunoblotting. Tubulin, loading control. The experiment was performed twice yielding similar results. e U-2 OS cells were transfected with siRNAs and levels of Tpr and GANP were assessed using immunofluorescence. Nuclei were stained with DAPI. The scale bar represents 7.5 µm. Data are representative of three independent experiments. f Expression of siRNA-resistant full-length Tpr-wt can rescue GANP protein level. U-2 OS cells were transfected with siRNA targeting endogenous Tpr (siTPR), and with siRNA-resistant Tpr-wt-GFP (TPR-wt). Levels of Tpr-wt-GFP and GANP were assessed using immunofluorescence. Nuclei, DAPI. The scale bar, 7.5 µm. Data are representative of three independent experiments. g Expressions of Tpr-N-terminal domain or Tpr-C-terminal domain are not able to rescue GANP protein level. U-2 OS cells were transfected with siRNA targeting endogenous Tpr (siTPR), and with Tpr-N-terminal-FLAG (TPR-N) or Tpr-C-terminal-FLAG (TPR-C) constructs. Levels of Tpr-FLAG and GANP were assessed using immunofluorescence. Nuclei, DAPI. The scale bar, 7.5 µm. Data are representative of three independent experiments. h Expression of siRNA-resistant full-length Tpr-NEBM (Nuclear Envelope Binding Mutant) is not able to rescue GANP protein level. U-2 OS cells were transfected with siRNA targeting endogenous Tpr (siTPR), and with siRNA-resistant Tpr-NEBM1-GFP (NEBM1) construct carrying L458P/M489P double amino acid substitution in Tpr nuclear envelope binding domain. Levels of Tpr-NEBM1-GFP and GANP were assessed using immunofluorescence. Nuclei, DAPI. The scale bar, 7.5 µm. Data are representative of three independent experiments. Source data for a–d are provided in the Source Data file.
Fig 2: Analysis of Tpr and GANP protein levels in human tumors.a Immunohistochemical analysis of Tpr and GANP protein levels on parallel sections of human tumors. Archival specimens from a cohort of 51 human serous ovarian carcinomas were stained with antibodies specific for Tpr and GANP using immunoperoxidase technique. Examples of immunohistochemical staining patterns seen among the ovarian tumors are shown. The scale bar represents 100 µm. b The summary of the scoring categories for the entire cohort of 51 human serous ovarian carcinomas analyzed.
Fig 3: GANP-depleted cells exhibit replication defects.a DNA fiber analysis of the replication fork speed and symmetry in nontreated conditions. Left: schematic of the CldU/IdU pulse-labeling protocol used. Middle: analysis of the replication fork speed. Total number of molecules analyzed: siCTRL (n = 362), siGANP (n = 407). The plot includes the mean ± SD. Statistical test: unpaired t-test with Welch’s correction, two-tailed. Right: analysis of the replication fork symmetry. The ratio between CldU/IdU was calculated from the same molecules used for the analysis of the replication fork speed (middle) (siCTRL n = 362, siGANP n = 407). Bounds of box are 25th–75th percentile, center shows the median, whiskers indicate the 10th–90th percentiles, data outside the range are drawn as individual dots. Statistical analysis: unpaired t-test with Welch’s correction, two-tailed. b DNA fiber analysis of the replication fork recovery upon HU removal. Left: schematic of the CldU/IdU pulse-labeling protocol used. Middle: length of the IdU tracks measured upon hydroxyurea removal. Total number of molecules analyzed: siCTRL (n = 190), siGANP (n = 181). The plot includes the mean ± SD. Statistical test: unpaired t-test with Welch’s correction, two-tailed. Right: analysis of the replication fork symmetry. The ratio between CldU/IdU was calculated from the same molecules used for the analysis of the length of the IdU tracks measured upon hydroxyurea removal (middle) (siCTRL n = 190, siGANP n = 181). Bounds of box are 25th–75th percentile, center shows the median, whiskers indicate the 10th–90th percentiles, data outside the range are drawn as individual dots. Statistical analysis: unpaired t-test with Welch’s correction, two-tailed.
Supplier Page from Abcam for Anti-GANP antibody