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. 2013 Apr 1;201(1):113–129. doi: 10.1083/jcb.201209098

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© 2013 Lin et al.

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Figure 3. — EPG-7 colocalizes with SQST-1 aggregates in autophagy mutants and directly interacts with SQST-1. (A) EPG-7 colocalizes with SQST-1 aggregates in epg-8 mutant embryos. Colocalization was defined as two aggregates showing >70% overlap in their fluorescence signals. Insets show a magnified view. (B) Percentage of EPG-7 aggregates colocalized with SQST-1 in indicated autophagy mutants. (C) A coimmunoprecipitation assay reveals that EPG-7 associates with SQST-1. (D) EPG-7 interacts with SQST-1 in a pull-down assay. (E) EPG-7 self-associates in a pull-down assay. (F) SDS-PAGE and blue-native PAGE of purified EPG-7(520–714) proteins. Lane M shows molecular weight marker. Lanes 1, 2, 3, and 4 contain 10, 5, 2.5, and 1.25 µg of proteins, respectively. (G) An EPG-7 fragment containing amino acids 531–631 self-interacts. (H and I) SQST-1 aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del 531–631)::gfp transgene. (H) DAPI image of the embryo shown in I. (J and K) EPG-7(del 531–631)::GFP is weakly expressed and diffusely localized in epg-7(tm2508) embryos. Its expression is not elevated in lgg-1(RNAi) animals. H–K show embryos carrying the same transgene. Images in J and K were taken using the same exposure time. (L and M) SQST-1 self-interacts (L) and the self-interaction is mediated by a fragment containing amino acids 26–181 (M). (N) Levels of SQST-1(del 26–181)::GFP are dramatically elevated in epg-7 and atg-3 mutants. (O and P) SQST-1(del 26–181)::GFP is weakly expressed and diffusely localized in sqst-1(ok2892) mutant embryos. (O) DIC image of the embryo shown in P. (Q and R) SQST-1(del 26–181)::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg-1 (Q) and epg-7 (R) mutants. Bars: (A, H–K, and O–R), 10 µm; (A, insets) 5 µm.