Rabbit anti gdf15

EndoC-betaH1 cells were preincubated with 10 µM imatinib or 0.5 mM metformin for 6 h. This was followed by treatment with or without the combination of palmitate (1.5 mM) and glucose (22 mM) for an additional 24 h, still in the presence of imatinib or metformin. After the exposure period, total protein lysates were prepared using SDS sample buffer, at pH 6.8, containing Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Samples were boiled for 5 min and separated on SDS-PAGE (4–20%, Bio-Rad, Hercules, CA, USA). Proteins were electrophoretically transferred to a Hybond PVDF membrane (GE Healthcare, Chicago, IL, USA) followed by blocking with 1% (w/v) BSA for 1 h. The membrane was then incubated with rabbit anti-GDF15 (1:1500, Abcam), mouse anti-beta-actin (1:1500, Santa Cruz, Santa Cruz, CA, USA), and mouse anti-alpha-tubulin (1:1500, Santa Cruz) antibodies overnight at 4 °C. Fluorescent anti-mouse (LI-COR Biosciences, Lincoln, NE, USA, IRDye) and anti-rabbit (LI-COR, IRDye) secondary antibodies, diluted to 1:15,000, were applied for 1 h at room temperature. Immunodetection was visualized and quantified using the LI-COR Odyssey Fc system (LI-COR).

Human islets were trypsinized so that a cell suspension was obtained, and then transferred to a clearly defined area of poly-lysine glass slides using a Cytospin 4 centrifuge (Thermo Scientific, Waltham, MA, USA). Thereafter cells were fixed with 4% (w/v) paraformaldehyde for 5–10 min at room temperature, blocked for 30 min using 2% BSA, permeabilized with 0.1% Tris-NaCl for 10 min, and stained overnight with the following primary antibodies: rabbit anti-GDF15 (Abcam, 1:250), guinea pig anti-insulin (Fitzgerald, Sudbury, MA, USA, 1:250), and mouse anti-glucagon (Thermo Fischer Scientific, 1:1000). All slides were washed with phosphate-buffered saline (PBS) and, prior to counterstaining, with diluted secondary antibodies: ALEXA Fluor 594 donkey anti-rabbit (Jackson, bar Harbor, ME, USA, 1:250), ALEXA Fluor 488 donkey anti-guinea pig (Jackson, 1:250), and ALEXA Fluor 594 goat anti-mouse (Life technologies, Carlsbad, CA, USA, 1:250) for 1 h at room temperature. Cells were washed with PBS and then mounted with Biotium’s (Freemont, CA, USA) EverBrite™ mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) for nuclei staining. Images were taken and analyzed with a Nikon Eclipse C1/TE2000-U microscopy (Nikon, Konan, Tokyo, Japan).

Cells were lysed in RIPA buffer (Cell Signaling; Danvers, MA) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Total protein extracts were run on SDS-PAGE and blotted onto nitrocellulose. Blots were probed overnight using the following antibodies from Cell Signaling: rabbit anti-phospho-IGF-1 receptor β (Tyr1131) (#3021, 1:200); rabbit anti-IGF-1 receptor β (#3018, 1:250); and rabbit-anti-N-Cadherin (#4061, 1:1000). Rabbit anti-GDF15 (#8479 1:200) was purchased from AbCam (Cambridge, MA). Mouse anti-E-Cadherin (#610181, 1:1000) was purchased from BD Biosciences (San Jose, CA). Mouse anti-vimentin (Sigma-Aldrich; V6630) was used at 1:1000. Mouse anti-β-actin monoclonal AC-15 (Sigma-Aldrich) was used at 1:10,000 as a loading control. All primary antibodies were diluted in 5% BSA/TBS-T. Goat anti-mouse secondary IRDye 800 antibody (#926-32210, 1:10,000) was purchased from Li-Cor Biosciences (Lincoln, NE). Goat anti-rabbit Alexa Fluor 680 secondary antibody (#1027681, 1:10,000) was purchased from Invitrogen (Grand Island, NY). Protein bands were detected using the Odyssey Imaging System (Li-Cor Biosciences, Lincoln, NE). Blots were repeated at least three times.