l685 458  (Tocris)

Journal: Cell Death Discovery

Article Title: TDP43 is a newly identified substrate for PS1, enhancing the expression of APP following cleavage

doi: 10.1038/s41420-025-02340-z

Figure Lengend Snippet: A PPI network interaction between PS1, APP, and TDP43. The representative image of the protein-protein interaction (PPI) network of PS1, APP, and TDP43, indicating potential interactions among these proteins. B Co-immunoprecipitation (Co-IP) of TDP43 and PS1. HeLa cells were co-transfected with TDP43 and PS1 plasmids. After sample collection, a Co-IP assay was performed using a PS1 antibody, followed by western blotting with a c-Myc antibody to detect the interaction between TDP43 and PS1. C Bioinformatics prediction of the binding site between TDP43 and PS1. The z-DOCK online platform predicted the optimal binding position between TDP43 and PS1. The surface interactions of the two proteins were subsequently analyzed using Discovery Studio. D Immunofluorescence co-localization of TDP43 and PS1. HeLa cells were transfected with LacZ as a blank control (Group 1) and co-transfected with TDP43 and PS1 (Group 2), as well as TDP43 and PS1 with the addition of γ-secretase inhibitor L685,458 1 h before transfection (Group 3). After 24 h, immunofluorescence analysis was performed to detect the interaction between TDP43 and PS1. DAPI was used to stain the cell nuclei, TDP43 was labeled with FITC to emit green fluorescence, and PS1 was labeled with PE to emit red fluorescence. The merge image shows the integration of all three channels. E Impact of the interaction of TDP43 and PS1 on the nuclear localization of TDP43. TDP43 and PS1 were overexpressed in NSC34 cells, after which the nucleus and cytosol were isolated. Finally, the expression of TDP43 in the nucleus and cytosol was detected by WB. F Grayscale values of endogenous TDP43. The grayscale values in the graphs were calculated using Image J software analysis, and data are presented as mean ± SD. Two-way ANOVA with post hoc Bonferroni correction, compared with nucleus. ( n = 3 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant).

Article Snippet: L685,458 was purchased from MedChem Express (NJ, United States) and Z-VAD-FMK from Selleck (TX, United States).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Western Blot, Binding Assay, Immunofluorescence, Control, Staining, Labeling, Fluorescence, Isolation, Expressing, Software

Journal: Cell Death Discovery

Article Title: TDP43 is a newly identified substrate for PS1, enhancing the expression of APP following cleavage

doi: 10.1038/s41420-025-02340-z

Figure Lengend Snippet: A PS1 Cleavage of TDP43. HeLa cells were seeded in 35-mm dishes for 24 h. Then, one group was transfected with LacZ as a control, the second with 4 μg of PS1 plasmid, and the remaining three groups were pre-treated for 1 h with z-VAD, L685,458, and with their combination before transfection. Endogenous TDP43 levels were detected by western Blot analysis (Panel 1). The membranes in the top panels were reprobed with GAPDH antibodies to indicate the relative sample loading (bottom panels). C Effect of PS1 mutants on TDP43. HeLa cells were transfected with LacZ, PS1, PS1 D257A, and PS1 D385A for 24 h. Then, western blotting was performed to assess the levels of TDP43, and PS1-cas to verify that PS1 had been transfected into cells (panel 2). E Immunofluorescence detection of TDP43 levels after PS1 overexpression and inhibitor treatment. HeLa cells were transfected with LacZ as a blank control (Lane 1), PS1 alone (Lane 2), and pre-treated with z-VAD and L685,458 for 1 h before PS1 transfection (Lane 3). After 24 h, TDP43 levels were detected by immunofluorescence analysis. The TDP43 fluorescence intensity was decreased in the PS1 group, while, it increased in the inhibitor-treated group. DAPI was used to stain the nuclei, and TDP43 was labeled with FITC to emit green fluorescence. The merge image shows the integration of both channels. B – F Grayscale values of endogenous TDP43. The grayscale values in the graphs were calculated using Image J software analysis, and data are the mean ± SD of at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant). The use of # indicates a comparison with the PS1+z-VAD + L685,458 group (# < 0.05, ## < 0.01, ### < 0.001, #### < 0.0001).

Article Snippet: L685,458 was purchased from MedChem Express (NJ, United States) and Z-VAD-FMK from Selleck (TX, United States).

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, Immunofluorescence, Over Expression, Fluorescence, Staining, Labeling, Software, Comparison