Fig 1: FIGURE 4: Model depicting the role of caspase 3 in proteostasis and mitochondrial function.In normal physiological state, moderate activation of caspase 3 leads to the disaggregation of TDP43 protein inhibiting its association with PHB2 leading to normal mitochondrial function. On the contrary, caspase 3 inhibition will facilitate the association of TDP43 with PHB2 which will lead to impaired mitochondrial function and can act as a driver for pathologies such as ALS.
Fig 2: FIGURE 2: Human Caspase 3 inhibits TDP43 protein aggregation.(A) Representative fluorescence image showing TDP43-RFP expression (red) in three yeast strains BY4741, ?ScMCA1 and hCasp3-GFP. (B) Western blot of TDP43-RFP protein was performed in three yeast strain BY4741, ?ScMCA1, and hCasp3-FP using anti-RFP antibody. The graph represents the difference in band intensities (normalized to control BY4741) across the three strains. Values are represented as mean ± SEM with p-value < 0.05. (C) Fluorescence image of ?ScMCA1 strain showing TDP-43-RFP (red) staining and vacuole stained with FM4-43FX (green). The graph shows the difference in percentage of vacuoles (=3 vacuoles per cell) across the three yeast strains. Values are represented as mean ± SEM with p-value < 0.05. (D) Mass Spectrometry (MS/MS) analysis of proteins immuno-precipitated with TDP43 using anti-RFP magnetic beads in hCasp3-GFP yeast strain with and without z-DEVD-FMK treatment. The graph represents proteins with high fold interaction (normalized to DMSO control) of TDP-43 post-z-DEVD-FMK treatment. (E) Representative co-immunoprecipitation blot of PHB2 with TDP43 using anti-TDP43 antibody in hCasp3-GFP yeast strain treated with DMSO, z-DEVD-FMK, and AC-1 performed in triplicates.
Fig 3: FIGURE 3: Caspase 3 inhibits TDP43 protein aggregation in skeletal muscle cells.(A) Representative immunofluorescence image of C2C12 cells treated with DMSO, z-DEVD-FMK and PAC1 showing protein PHB2 (green) and TDP43 (red) and their colocalization (yellow). The graph represents the percentage of TDP43 inclusions in C2C12 cells treated with DMSO, z-DEVD-FMK, and PAC1. The experiments were performed in triplicates and the values are represented as mean ± SEM with p-value < 0.05. (B) Representative immunofluorescence image of human primary myoblast treated with DMSO and PAC1 showing protein PHB2 (green) and TDP43 (red) and their colocalization (yellow). The graph represents the percentage of TDP43 inclusions in n primary myoblast cells treated with DMSO, z-DEVD-FMK, and PAC1. The experiments were performed in triplicates and the values are represented as mean ± SEM with p-value < 0.05 (C) Representative fluorescence image of C2C12 cells treated with DMSO and z-DEVD-FMK stained with MitoTracker Red CMXRos for assessment of mitochondrial function. The graph represents difference in mean fluorescence intensity of MitoTracker Red CMXRos (red) in C2C12 cells treated with DMSO and z-DEVD-FMK. The experiments were performed in triplicate with values represented as mean ± SEM with p-value < 0.05.
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