pCDNA3 hemagglutinin (HA)–Trim32 had been previously described (10). The HA-Trim32 mutants that correspond to human complementary DNA (cDNA) pathogenic Trim32 mutations were engineered with the QuickChange Site-Directed Mutagenesis Kit (StrateGene).

All retroviral constructs were based on a modified version of a pLPCX vector (Clontech) (32). pLPCX plasmids encoding FLAG- and MYC-WT AMBRA1, MYC-AMBRA1 mutants (amino acids 1 to 532, 533 to 751, and 761 to 1269), and FLAG–BECLIN 1 were previously described (3032). pLPCX FLAG-TRIM32 and pLPCX TRIM32 were obtained by subcloning from pCDNA3 HA-TRIM32. pLPCX FLAG-TRIM32 deletion mutant RING/B-box (amino acids 1 to 136), coiled-coil domain (amino acids 136 to 326), NHL repeats (amino acids 327 to 653), and ΔNHL (amino acids 1 to 325) were created using the appropriate oligonucleotides and amplification, followed by in-frame insertion into the pLPCX FLAG vector. pCDNA3 HA-Trim32 C39S was generated by site-specific mutagenesis; pLPCX FLAG-TRIM32 C39S was obtained by subcloning from pCDNA3 HA-TRIM32 C39S. pLPCX FLAG-ULK1 and pLPCX MYC-ULK1 was obtained by subcloning from pcDNA3 Myc-tag ULK1 (31). pLPCX FLAG-ULK1 deletion mutants (amino acids 1 to 828 and 829 to 1050) were created using appropriate oligonucleotides and amplification, followed by in-frame insertion into the pLPCX FLAG vector. pRK5 HA-UBIQUITIN was obtained from Addgene (#17608). A CRISPR-Cas9 All-in-One lentiviral vector specific for human TRIM32 and murine TRIM32 [hTRIM32 single-guide RNA (sgRNA), K2465605; mTRIM32 sgRNA, K3452705] and control sgRNA (scramble sgRNA, K010) were purchased from ABMgood.

For stable murine Trim32 mRNA interference, two lentiviral Trim32 mRNA–targeting pLKO.1 plasmids were used (TRCN0000040831 and TRCN0000040832; Sigma-Aldrich). For stable murine Ambra1 mRNA interference, two lentiviral Ambra1 mRNA–targeting pLKO.1 plasmids were used (TRCN0000189905 and TRCN0000189940; Sigma-Aldrich). For stable human AMBRA1 mRNA interference, a lentiviral AMBRA1 mRNA–targeting pLKO.1 plasmid was used (TRCN0000168652; Sigma-Aldrich). For stable murine Nedd4L mRNA interference, a lentiviral Nedd4L mRNA–targeting pLKO.1 plasmid was used (TRCN0000086870; Sigma-Aldrich). A pLKO.1 containing a nonmammalian shRNA was used as a negative control (Sigma-Aldrich).

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