Ab92517

The following antibodies were used: anti-pancreatic alpha amylase (ab21156, Abcam, Cambridge, U.K.), anti-insulin (sc-7839, Santa Cruz Biotechnology, Dallas, TX, USA), anti-somatostatin 28 (ab22682, Abcam), anti-glucagon (ab92517, Abcam), and anti-GFP (Rockland Immunochemicals, Gilbertsville, PA, USA). Rabbit anti-CK19 antibodies were a gift from Dr. Atsushi Miyajima (The university of Tokyo) [41 (link),42 (link)]. PDL-administered pancreas or pancreatic organoids from Ngn3-GFP mice (n = 3, respectively) were fixed in 4% paraformaldehyde in phosphate-buffered saline and embedded in O.C.T. compound (Sakura Finetek, Torrance, CA, USA). Frozen sections (12 µm) were prepared and incubated in 0.3% Triton X-100 and 2% donkey serum for 1 h, followed by incubation with the primary antibodies overnight at 4 °C. Next, the sections were incubated with secondary antibodies (Alexa Fluor 555 or 594 IgG; Invitrogen, Carlsbad, CA, USA) and counterstained with DAPI (Nacalai Tesque, Kyoto, Japan).

Diabetes-mimicking αTC1-6 cells were generated as mentioned above and cultured on coverslips. Experiments were done in the presence or absence of lysosomal inhibitor, BFA1, in serum-free DMEM containing 16.7 mM glucose and 0.1% BSA for 2 h. Cells were then washed 3X in PBS, fixed in 2% paraformaldehyde for 30 min, washed 5X in PBS, and incubated with blocking buffer (2% BSA in PBS containing 0.05% Tween 20) for 1 h. Cells were then incubated with appropriate primary antibodies prepared in blocking buffer against glucagon (mouse anti-glucagon antibody, Cat # ab10988, Abcam; 1:1000 or rabbit anti-glucagon antibody, Cat# ab92517, Abcam; 1:25), Stathmin-2 (goat anti-SCG10 antibody, Cat # ab115513, Abcam; 1:250), or Lamp1 (mouse anti- Lamp1 antibody, Cat # ab25630, Abcam; 1:50). Following an overnight incubation at 4°C, coverslips were washed with PBS, and incubated for 2 h in the dark at RT with appropriate secondary antibodies (donkey anti-mouse IgG Alexa Fluor 488, Cat# A-21202, Molecular Probes; donkey anti goat IgG Alexa Fluor 555, Cat# A-21432, Molecular Probes; donkey anti-rabbit IgG Alexa Fluor 647, Cat# A-31573, Molecular Probes). Then, coverslips were washed with PBS, stained with DAPI, and mounted on glass slides using ProLong antifade mountant. Image acquisition and analysis was done as described above. Experiments were repeated four times using freshly thawed cells.

Mouse monoclonal antibodies against insulin (clone K36AC10, I2018, for immunostaining of paraffin sections), FLAG (M2, F1804), β-tubulin (DM1A, T9026), and glucagon (K79bB10, G2654) were from Sigma-Aldrich. Rabbit polyclonal antibodies against glucagon (ab92517), NNAT (ab27266), VAPB (ab72470), and ribophorin 1 (ab198508) and mouse monoclonal antibodies against PDI (RL90, ab2792) were from Abcam. Mouse anti-insulin (L6B10, 8138, for immunoblotting/immunofluorescence), anti–c-Myc (9B11, 2276), and anti-IRE1α (14C10, 3294) were purchased from Cell Signaling. MG132 was purchased from Sigma-Aldrich.

To confirm the 5-HT_2CR protein expression in pancreatic samples and MIN6 cells, immunofluorescence was performed as described previously (Schultz et al., 2020 (link)). Mouse pancreases were fixed in 4% paraformaldehyde for 24 h at 4°C, embedded in paraffin, and sectioned. The slices were blocked with 5% goat serum plus in PBS for 1 h and incubated with a mouse anti-5-HT_2CR monoclonal antibody (1:100, sc-17797, Santa Cruz) and rabbit anti-insulin antibody (1:250, ab181547 Abcam, Cambridge) or rabbit anti-glucagon (1:250, ab92517, Abcam) and then incubated with Alexa Fluor 488–conjugated donkey anti-mouse IgG (1:1000, A21202, Thermo Fisher) and Alexa Fluor 594–conjugated anti-rabbit IgG (1:1000, A21207, Thermo Fisher) for 1 h at RT. To stain nuclear DNA, the cells were treated with DAPI (ab104139, Abcam) for 5 min at RT. Negative control included the absence of primary antibodies. Images were taken on a Leica DM2500 microscope.
MIN6 cells were cultured on a glass slide and fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100 for 30 min, and blocked with 5% BSA for 30 min at room temperature, then cells were incubated with primary antibodies, the second antibodies and DAPI as above. Images were visualized by an Olympus FV1200 confocal laser scanning microscope system.