Skip to main content
. 2021 Jun 24;12:3937. doi: 10.1038/s41467-021-24224-3

Fig. 4. Tpr maintains normal protein levels of GANP.

Fig. 4

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.