Fig. 6.

Tsg101 promotes p62 aggregation and Keap1 recruitment to the Tsg101-p62 complex. (A) Diagram showing the p62-Keap1-Nrf2 signal pathway. Aggregation of p62 increases affinity of p62 to Keap1 and thereby, releasing Nrf2 from the Keap1-inhibition and translocating to the nucleus to initiate transcription of genes involved in anti-oxidant response. (B) Representative immunoblots and quantification analysis showing the expression levels of Tsg101, Keap1, p62 and S351-p62 in WT- and TG-hearts. *, p < 0.05 vs. WT. (C/D) Representative Western-blots showing aggregates of p62 and Tsg101 in the soluble and insoluble extracts from WT- and TG-hearts. n = 4 for each group. (E) Co-immunoprecipitations (Co-IP) with anti-p62 antibody and immunoblotting with Tsg101-antibody, using insoluble fractions of WT-and TG-hearts. Insoluble fractions of TG hearts were used as positive controls, and immunoprecipitates with IgG as negative control. HC stands for non-specific IgG heavy-chain bands. (F) Co-IP with Tsg101-antibody and immunoblotting with p62-antibody, using insoluble fractions of WT and TG hearts. Insoluble fractions of TG hearts were used as positive controls, and immunoprecipitates with IgG as negative control. (G) Co-IP with anti-p62 antibody and immunoblotting with Tsg101-antibody, using the homogenates from pre- and post-I/R wild-type hearts. (H) Co-IP with Tsg101-antibody and immunoblotting with p62-antibody, using the homogenates from pre- and post-I/R wild-type hearts. Immunoprecipitates with IgG as negative controls. (I) Representative images showing immunofluorescence staining with antibodies to p62- (red) and Keap1 (purple) in Ad.GFP-Tsg101 (green)-infected neonatal rat cardiomyocytes (NRCMs). Similar results were observed in three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)