Ab181547

Insulin samples (2 μL) were spotted onto a nitrocellulose membrane (0.22 μm, GE Healthcare Life Sciences, Buckinghamshire, UK). The membrane was blocked with 5% skim milk in 5% BSA/Tris-buffered saline (TBS) containing 0.01% Tween 20 for 1 h at room temperature. Subsequently, the membrane was incubated with primary anti-insulin antibody (1:3000, ab181547, Abcam, Cambridge, MA, USA) and anti-oligomer antibody (1:3000, A11, Invitrogen, Carlsbad, CA, USA) for 24 h. The following day, the membrane was incubated with the secondary anti-rabbit IgG antibody (1:10,000; R&D Systems, Minneapolis, MN, USA) for 1 h, as described^{10 (link)}. After washing with TBST, proteins were visualized using an ECL blotting detection kit (Bio-Rad) according to the manufacturer’s instructions. Luminescence was detected with a Las 4000 mini luminescent image analyzer (Fujifilm, Tokyo, Japan) using the Image Reader Las 4000 software. ImageJ was used to determine the intensity of each dot, which was used to quantify the amount of antibodies on the membrane.

In order to study the pancrease morphology, 8 pancreases from each group of rats were removed and weighed, excised fat and lymphatic tissues for serial section for serial section (19 (link), 20 (link)). β-cell mass was measured by point-counting stereology according to the previous research (21 (link)). After fixing in cold acetone, tissues were incubated with primary antibodies (GPER, ab39742; GLUT2, ab54460, insulin, ab181547; glucagon, ab10988; 1: 200, respectively, Abcam, USA) at 4°C overnight. Then tissues were washed and incubated with the secondary antibody (Alexa Fluor^® 488 Conjugate anti-rabbit IgG and Alexa Fluor^® 555 Conjugate anti-rabbit IgG, 1:1,000, #4412 and #4413, Cell Signaling Technology, USA) at room temperature for 1 h. Sections were washed again and then DAPI stained (#4083, Cell Signaling Technology, USA) at room temperature for 10 min. Images of the sections were taken under an Olympus fluorescence confocal microscope (FV-1000, Japan).

The anti-USF1 antibody is a rabbit polyclonal IgG directed against the C-terminus of USF1 (c-20, sc-229) or a rabbit polyclonal IgG directed against the amino acids 75–160 of USF1 (H-86, sc-8983), whereas the anti-USF2 antibody is a rabbit polyclonal IgG directed against the C-terminus of USF2 (c-20, sc-862). HA-tagged dominant negative USF mutant was detected with the HA specific mouse monoclonal antibody 12CA5 from Roche Diagnostics (Mannheim, Germany). PDX-1 was identified with a polyclonal antiserum generated by immunizing rabbits with recombinant mouse PDX-1^{20 (link),61} . The mouse monoclonal antibody against CK2β (E-9, sc-46666) was purchased from Santa Cruz (Biotechnology Inc., Heidelberg, Germany). Detection of CK2α was performed by using the mouse monoclonal antibody 1 A5^{62 (link)}. The mouse monoclonal antibody FLAG M2 (F1804), the β-actin and the α-tubulin (clone DM1A) antibody were from Sigma-Aldrich (Munich, Germany). Rabbit polyclonal serum #36 against nucleolin was prepared in our laboratory^{63 (link)}. Insulin was detected by the monoclonal rabbit antibody AB181547 from abcam (Cambridge, UK).

Paraffin embedded tissues were sectioned at 5–6 μm and OCT tissues were cryosectioned (Thermoscientific, UK) at 8–10 μm thickness between -14 to -30°C. All tissues were haematoxylin and eosin (H&E) stained, gut and liver tissue sections were also stained with Gömöri’s Trichrome using previously described methods [12 (link)]. Prior to immunofluorescence (IF) staining, antigen retrieval (10 mM citrate buffer, pH 6) was performed and the sections were avidin/biotin blocked (Vectorlabs, UK). Briefly, pancreatic insulin and glucagon were stained with rabbit anti-insulin (1/200 dilution: Catalogue number Ab181547 Abcam, UK) and mouse anti-glucagon antibodies (1/200 dilution: Catalogue number Ab10988 Abcam, UK) overnight, followed by goat anti-rabbit IgG Alexafluor 488 (1/400 dilution: Abcam, UK) and goat anti-mouse IgG Alexafluor 647 (1/400 dilution: Catalogue A28181 Invitrogen, UK). IL-17 was detected using goat anti-mouse IL-17 (1/100 dilution: Catalogue number Af-421-na R&D Systems, UK) followed by biotinylated rabbit anti-goat IgG (1/200 dilution: Catalogue number 31732 Invitrogen, UK) with streptavidin Alexafluor 647 (1/200: dilution Catalogue number S21374 Invitrogen, UK). IF sections were mounted with Vectashield with DAPI (Vectorlabs, UK) and images were acquired with an EVOS FL Auto 2 system (Thermofisher, UK).

To estimate histological changes, embedded pancreatic tissues were sectioned to a thickness of 8 μm for hematoxylin/eosin (HE) staining (G1121; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), Masson’s trichrome staining (G1346; Beijing Solarbio Science s& Technology Co., Ltd., Beijing, China), and immunofluorescent staining. Mouse anti-insulin (rat, 1:500, ab181547; Abcam, Waltham, MA, USA), rabbit anti-glucagon (rabbit, 1:150, 15954-1-AP; Proteintech, Rosemont, IL, USA), and the corresponding secondary antibodies (donkey anti-rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555; donkey anti-mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488(REF.A21202); Life Technologies, Carlsbad, CA, USA) were used to detect alpha and beta islet cells. As described in Chansela et al.’s study, the islets could be categorized as small (<10,000 μm²), medium (10,000–50,000 μm²), large (50,000–100,000 μm²), and extra large (>100,000 μm²) on the basis of the total area of islets [43 (link)]. The islet density is calculated as follows: the number of each islet size/total number of islets ×100 [43 (link)].