Total protein was extracted from 50–500 islets isolated from two or three control or β-cell-specific VPS13C knockout (βVps13cKO) mice (males and females) in ice-cold RIPA buffer [1% (vol/vol) Triton X-100, 1% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) SDS, 0.15 M NaCl, 50 mM Tris, pH 8.0] containing a 2× concentration of Complete, EDTA-free protease inhibitor cocktail (Roche, Burgess Hill, UK). The samples were incubated in RIPA on ice for 10 min and then freeze-thawed twice to ensure release of proteins. Samples were clarified by centrifuging at 16,000 g for 10 min at 4°C, and then total protein content was quantified using a BCA protein assay kit (Pierce, ThermoScientific). Total protein (5 μg) was added to SDS sample buffer [0.5 M Tris·HCl, pH 6.8, 2% (wt/vol) SDS, 5% (wt/vol) glycerol, 0.6 M DTT, and 0.2 mg bromophenol blue] and incubated at room temperature for 30 min. Samples were then electrophoresed on a 4–10% discontinuous gradient gel alongside a HiMark Protein Standard (Novex; ThermoScientific) and transferred onto a nitrocellulose membrane overnight. Membranes were blocked with 5% milk and incubated with primary antibodies (rabbit anti-VPS13C, 1:2,000, described above, or goat anti-EEA1, Santa Cruz, TX, 1:2,000) overnight with agitation at 4°C. Membranes were then washed three times in PBS-Tween 20 (0.2% vol/vol) and incubated with horseradish peroxidase-conjugated antibodies for 1 h at room temperature. Following three washes in PBS-Tween 20, proteins were visualized with ECL reagent and X-ray film (Amersham, GE Healthcare Life Sciences).