Luminata western hrp substrate

Following stimulation, fibroblasts were lysed with RIPA buffer (Cell Signaling Technology) containing phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA). Nuclear lysates were prepared using a LysoPure™ Nuclear and Cytoplasmic Extractor Kit (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) containing protease inhibitor cocktail Set III (FUJIFILM Wako Pure Chemical Corporation). Protein levels were quantified using precision red advanced protein assay reagent #2 (Cytoskeleton, Denver, CO, USA). Equal amounts of total protein were separated on 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred onto an immobilon-P transfer membrane (Millipore, Billerica, MA, USA) by a PoweredBLOT 2M system (ATTO, Tokyo, Japan). The membranes were blocked with ImmunoBlock (KAC, Hyogo, Japan) for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight. The membranes were washed with Tris-buffered saline with 0.1% Tween 20 and incubated with HRP-conjugated secondary antibodies. After being washed, the membranes were developed with Luminata Western HRP substrate (Millipore, Billerica, MA, USA), and the bands were detected with ImageQuant™ LAS 4000 (FUJIFILM Wako Pure Chemical Corporation). The intensity of the bands was quantified using ImageJ software (NIH, Bethesda, MD, USA).

Rabbit polyclonal antibody to insulin degrading enzyme (IDE, Cat. ab32216) and mouse monoclonal to β-Actin (Cat. ab6276) were from Abcam (1 Kendall Square, Suite B2304 Cambridge, MA 02139–1517, USA). Secondary antibodies include ECL Anti-rabbit IgG horseradish peroxidase linked whole antibody (from donkey, Cat. NA934V, and ECL Anti-mouse IgG horseradish peroxidase linked whole antibody (from sheep, Cat. NA931V, GE Healthcare UK limited, Little Chalfont Buckinghamshire, HP7 9NA UK). Immobilon-P^SQ PVDF Transfer Membranes (Cat. ISEQ 10100) and Chemiluminescence reagent Luminata Western HRP Substrate (Cat. WBLUF0100) were from Millipore (Billerica, MA 01821). The X-ray film was from Phenix Research (Cat. F-BX810, Candler, NC, USA).

Frozen tissue was homogenized with Quiagen Tissue Ruptur in 1.5 ml of cold 20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, 70 mM sucrose and centrifuged at 1.500×g for 5 min at 4°C. Samples were then analysed by SDS gel electrophoresis (Novex, Life technologies, Australia) and electroblotted to Hybond Nitrocellulose membranes (Amersham Pharmacia Biotech, Bucks, UK). Membranes were then probed with primary antibodies to GLUT1 (ab652, Abcam, Cambridge, UK), SGLT2 (sc98975, Santa Cruz, USA) and actin (Santa Cruz, USA). Proteins were visualized using Luminata Western HRP Substrate (Millipore) in a LAS 4000 image reader (GE Healthcare Life Sciences). Analysis was performed using Image J software (NIH, USA).

OGCs in different groups were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer (Solarbio) containing 1 mM phenylmethanesulfonyl fluoride (PMSF) (Solarbio) at 4 °C for 30 min. The protein concentration was determined with a BCA Protein Assay Kit. Up to 50 µg of protein was electrophoresed on 12% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore), which were blocked with 5% nonfat dry milk (BD Biosciences) for 1 h at room temperature. The membranes were then blotted with primary antibodies at 4 °C overnight. The following primary antibodies were used: β-actin (1:1,000, Proteintech, Rosemont, IL, USA), B cell lymphoma 2 protein (Bcl-2, 1:1,000, Cell Signaling Technology), Bcl-2-associated X protein (Bax, 1:2,000, Abcam), caspase-3 (1:1000, Cell Signaling Technology), cleaved caspase-3 (1:1,000, Cell Signaling Technology) and cleaved poly-ADP-ribose polymerase (cleaved PARP, 1:250, Abcam). Then, the PVDF membrane was washed 3 times with Tris-buffered saline/Tween (Solarbio) and incubated with HRP-conjugated secondary antibody (1:10,000, ZSGB-BIO) for 70 min at room temperature. Detection was performed using Luminata western HRP substrate (Millipore). The results were obtained with a LI-COR 3600 instrument (LI-COR Biosciences, Lincoln, NE, USA) and analysed with an Image Studio Digits Version 4.0 system; n = 5.