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Fig. 6 | Journal of Biomedical Science

Fig. 6

From: N-terminal functional domain of Gasdermin A3 regulates mitochondrial homeostasis via mitochondrial targeting

Fig. 6

N-Gsdma3 associates with Hsp90 and the mitochondrial chaperone Trap1. (a) Proteomics analysis. Anti-flag immunoprecipitation followed by SDS-PAGE and silver staining of transfected HEK293T cells. Unique gel bands (Gel1 and Gel2) revealed in the N-Gsdma3 immunoprecipitate were excised out and processed for proteomics analysis. (b, c) Reciprocal co-immunoprecipitation of Hsp90 and N-Gsdma3. (d) Reciprocal co-immunoprecipitation of N-Gsdma3 and Trap1. (e, f) Quantification results for MitoSox Red staining. The bar graphs depict the mitochondrial ROS levels (mean + s.d., n = 3 in duplicates) in radicicol (750 nM)-treated transfected cells relative to those in vehicle-treated transfected cells. (g, h) Quantification results for 7AAD and DiIC1(5) staining. The bar graphs represent fractions of dead (7AAD+) cells and mitochondrial membrane potential depolarization (DiIC1(5)low) cells (mean + s.d., n = 3 in duplicates) in radicicol (750 nM)-treated transfected cells relative to those in vehicle-treated transfected cells. pIRES vector-transfected cells were used as the staining baseline. ***, P < 0.001. No apparent cell death was induced by 750 nM radicicol treatment of HEK293T cells for 20 hours. (i) Co-immunoprecipitation of N-Gsdma3 and Gsdma3Rim3 with Hsp90/Hsp70 in transfected HEK293T cells in the absence or presence of 750 nM radicicol. (j) Immunoblot analysis of AKT protein in HEK293T cells after treatment with radicicol (250, 750, 1000 nM) for 20 hours. β-actin was used as a loading control. Relative densitometry intensities are indicated above the protein bands. (k) Reciprocal co-immunoprecipitation of Tom70 and N-Gsdma3 in the absence or presence of 750 nM radicicol (l) Immunoblot analysis of fractionated mitochondria from transfected HEK293T cells in the absence or presence of 750 nM radicicol either left untreated (-) or treated with 5 μg/ml proteinase K (+PK). Tom20 was used as an indicator of PK treatment; Cox4 was used as the loading control. (m) Quantification of the PK protection assay. The bar graph depicts the densitometry levels of N-Gsdma3 in the radicicol-treated preparation relative to the untreated preparation (mean + s.d., n = 3)

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