293 T cells were transiently transfected with expression vectors using the calcium phosphate method, as previously described (32). For retroviral production, packaging cells [293 gp/bsr (gag-pol/blasticidin S-resistant gene)] were cotransfected with 15 μg of retroviral vectors and 5 μg of pCMV-VSV-G using the calcium phosphate method. For lentiviral production, 293 T cells were cotransfected with 10 μg of lentiviral vectors, 2.5 μg of pCMV-VSV-G, and 7.5 μg of psPAX2 plasmid by using the calcium phosphate method. After 48 hours, the supernatant containing the retroviral or lentiviral particles was recovered, ultracentrifuged at 19,800 rpm on an SW28 rotor for 2 hours, and resuspended in phosphate-buffered saline (PBS) (500 μl for 20 ml of supernatant). Cells were infected with 80 μl of viral suspension in a medium supplemented with polybrene (4 μg/ml) for 8 hours. Two consecutive rounds of infections were performed to improve efficiency.

To establish Trim32 KO cells for mutant complementation assays, C2.7 cells were transiently transfected with CRISPR-Cas9 vectors by Lipofectamine LTX and Plus Reagent (Invitrogen, 15338-100), as indicated by the supplier. Transduced cells were incubated with puromycin dihydrochloride (2.5 μg/μl; Santa Cruz Biotechnology, sc-108071) for only 48 hours to select transfected cells, where inactivation of Trim32 CRISPR-Cas9 has occurred, and then cultured in the absence of puromycin to avoid selecting cells with a stably integrated CRISPR-Cas9 vector.

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