FIG. 8.

FIG. 8.

Srag interacts with Becn1. (A) Colocalization analysis between Srag and Becn1. HeLa cells were transiently cotransfected with GFP-Srag and mCherry-Becn1, followed by confocal microscopy. Colocalizing structures were indicated in yellow (merge). Arrows indicated the signals overlapped between Srag and Becn1. The nuclei were revealed using Hoechst fluorescence. Scale bar: 5 μm. (B) Co-IP analysis between endogenous Srag and Becn1 in testis of the adult Monopterus. The lysates were immunoprecipitated with anti-Srag, anti-Becn1 antibody, or negative serum, respectively, followed by immunoblotting with the anti-Becn1 or anti-Srag antibody, respectively. Arrows indicated the Co-IP band. (C) Schematic diagram of the Monopterus Becn1 wild-type and various deletions. The conserved domains (BH3, CCD, ECD, and C-terminal) are indicated in boxes. (D) Co-IP between Srag and deletion mutants of Becn1. 293T cells were transiently cotransfected with 3xFLAG-Srag and MYC-Becn1, MYC-Becn1-BH3Δ, MYC-Becn1-CCDΔ, or MYC-Becn1-ECDΔ. After transfection for 48 h, the whole-cell lysates were extracted for Co-IP with anti-FLAG or anti-MYC antibody, and anti-MYC or anti-FLAG antibody was used for western blot, respectively. The cell lysates were examined by western blotting using the anti-MYC or anti-FLAG antibody (input). (E) Schematic diagram of the Monopterus Becn1 truncated mutants. The conserved domains (BH3, CCD, ECD, and C-terminal) are indicated in boxes. (F) Co-IP analysis showed that Srag interacted with the C terminus of Becn1. 3xFLAG-Srag was cotransfected with MYC-Becn1 1–137, MYC-Becn1 138–352, MYC-Becn1 353–447, and MYC-Becn1 138–447 into 293T cells. The cell lysates were examined by western blotting using the anti-MYC or anti-FLAG antibody (input). The lysates were immunoprecipitated with the anti-FLAG or anti-MYC antibody, followed by immunoblotting with anti-MYC and anti-FLAG antibody, respectively. 3xFLAG-Srag can interact with MYC-Becn1 353–447 and MYC-Becn1 138–447. (G) Sequence alignments of the conserved C-terminal (pink) of BECN1 in human, Monopterus, and zebrafish. (H) Sox9–Srag–Becn1 pathway in autophagy regulation. srag promoter activity is activated by its transcription factor Sox9, which is bound to the srag promoter region in vivo. Srag interacts with the C-terminal of Becn1 to promote autophagy via PI3K complex upon starvation induction.