Pcaggs gfp plasmid

5 × 10⁵ cells/well in 6-well plates were co-transfected with wild-type (WT) or mutant FLAG-tagged RIMS2 plasmids (1.6 μg; Genscript) and the pCAGGS-GFP plasmid (400 ng; Clontech) with the FuGene HD transfection reagent according to the manufacturer’s protocol (Promega). Proteins were prepared with radioimmunoprecipitation assay (RIPA) lysis buffer (Life Technologies Thermo Fisher Scientific). 50 μg of total proteins were resolved by Mini-ProteanTGX Stain Free 4%–15% gel according to the recommendations of the supplier (BioRad). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane via an RTA Transfer Kit (BioRad), and the membrane was probed with the following primary antibodies: polyclonal goat IgG anti-DDDDK tag (1:5,000; Abcam) and monoclonal mouse IgG1κ anti-GFP (1:2,000; Sigma Aldrich). Rabbit anti-goat IgG-HRP (1:2,000; Abcam) and rabbit anti-mouse IgG-HRP (1:2,000; Abcam) were used as secondary antibodies. Blots were developed with the Clarity Western ECL and ChemiDoc XRS+ Imaging System (BioRad). Immunoblot images were acquired and analyzed via Image Lab Software 3.0.1 build 18 (BioRad). The abundance of FLAG relative to GFP was estimated by densitometry with the Image Lab Software 3.0.1 build 18. A Student’s t test was performed.

Clone B1 derived from Mouse insulinoma pancreatic 6 β-cells (MIN6 B1) were seeded at 3 × 10⁶ cells/well in 6-well plates and co-transfected with WT or mutant FLAG-tagged RIMS2 plasmids (2 μg, Genscript) and the pCAGGS-GFP plasmid (1 μg; Clontech) via the FuGene HD transfection reagent according to the manufacturer’s protocol (Promega). GFP-expressing cells from cell pools were sorted on a BD FACSAria II (BD Biosciences) with special order research products (SORP) program. GFP-MIN6 B1 cells were seeded at 5 × 10⁴ cells/well in 96-well plates. After 1 h of DMEM Glutamax I medium (5 mM glucose), the medium was replaced by DMEM Glutamax I medium (25 mM glucose). Concentration of insulin in the medium was determined with the Insulin Mouse ELISA Kit (Invitrogen Thermo Fisher Scientific) after 25 min of incubation. Absorbance was measured immediately at 450 nm and 550 nm by a VICTOR X4 2030 multilabel plate reader (PerkinElmer). Using Prism6 software, we determined the significance of variations among samples via a one-way ANOVA with a post hoc Tukey’s test.