INS 832/13 cells lacking TRAPα were generated using CRISPR-Cas9–mediated genome editing (49). Single-guide RNAs were designed using guide design resources available on the Zhang Lab website ( Oligonucleotides corresponding to 5′-CACCGTCTGCACTCGGTGAAGCTTC-3′ 20-nucleotide guide sequences were annealed and ligated into the pSpCas9(BB)-2A-Puro (PX462) v2.0 vector as described (49). This plasmid was transfected into INS 832/13 cells using Lipofectamine 2000 (Thermo Fisher Scientific). Forty-eight hours after transfection, cells were plated and cultured with RPMI 1640 growth medium containing puromycin (1 μg/ml). Puromycin-resistant clones were screened for loss of TRAPα (“TRAPα KO”) by immunoblotting with anti-TRAPα antibodies. To reexpress TRAPα, TRAPα KO cells were transiently transfected with a plasmid encoding Myc-tagged human TRAPα. Forty-eight hours after transfection, insulin content was assayed by immunofluorescence. To reduce TRAPα expression by RNAi, INS 832/13 cells were transfected with 20 nM TRAPα targeting small interfering RNA (siRNA; Thermo Fisher Scientific, assay ID 219062) or negative control siRNA (Thermo Fisher Scientific, AM4611) using Lipofectamine RNAiMAX (Invitrogen). Forty-eight hours after transfection, knockdown efficiency was evaluated by immunoblotting using anti-TRAPα antibodies.

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