Las 3000 image analyzer

Lysates from 1~2 x 10⁶ cells were prepared in SDS-PAGE sample buffer or NP-40 lysis buffer (50 mM HEPES, pH 7.4, 10% glycerol, 1% NP-40, 4 mM EDTA, 100 mM NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 mM PMSF, and 1 mM Na₃VO₄). Immunoprecipitation was performed at 4°C for 5 hours using protein G pre-coated with antibody. Immunoprecipitates were dissolved in SDS-PAGE sample buffer, subjected to SDS-PAGE, and electro-transferred onto PVDF membranes. Immunodetection was performed by ECL (PerkinElmer, Waltham, MA). Sequential re-probing of membranes was performed after the complete removal of primary and secondary antibodies in stripping buffer, or by inactivation with peroxidase in 0.1% NaN₃. Results were analyzed with an LAS-3000 image analyzer with Science Lab software (Fujifilm, Tokyo, Japan) or with a c-Digit imaging system with Image Studio Digit software (Licor Biosciences, Lincoln, NE).

Mice liver tissues were homogenized in RIPA buffer (Abcam, USA) containing protease and phosphatase inhibitors (Abcam, USA). The supernatant was isolated, and total protein concentrations was measured using a BCA kit (Thermo Scientific, USA). Denatured proteins were separated in 10% SDS-PAGE gel, then transferred to polyvinylidene fluoride (PVDF) membrane (GE Healthcare Life Science, Germany) using the Mini-PROTEAN Tetra Cell System (BioRad Laboratories Inc., CA, USA). The membranes were blocked by 5% skim milk with TBST and Tris-buffered saline, then washed with Tween 20 for 1 h and treated with primary antibody (1:10,000) overnight at 4°C. Samples were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000, beta actin manufactured by Santa Cruz, USA; AMPK, phosphorylated-AMPK and GLUT2 manufactured by Cell Signaling, USA) for 1 h. Detailed information regarding the antibodies is shown in Table S3. Finally, the band on membranes were detected using SUPEX ECL solution and photographed using a FUJIFILM LAS3000 Image Analyzer (FUJI, Japan).

Samples from cell lysates (20 µg total protein), precipitation of surface proteins or vesicle preparations (see below) were heated in reducing or non-reducing SDS-sample buffer (250 mM Tris HCl (pH 6.8), 50% (w/v) glycerol, 10% (w/v) SDS, 0.1% bromophenol blue with or without 5% β-mercaptoethanol) and subjected to SDS–polyacrylamide gel electrophoresis using 10% Tris–glycine gels. Proteins were transferred onto polyvinylidene difluoride membranes (Hybond-P, Amersham). Membranes were blocked with 5% (w/v) non-fat dry milk in PBS with 0.05% Tween for 1 h and probed with primary antibodies against ADAM10 (0.5 µg/ml) or marker proteins for 1 h at room temperature or overnight at 4 °C followed by incubation with HRP-coupled secondary antibodies (diluted 1:30,000) for 1 h. After addition of enhanced chemiluminescence substrate (ECL Prime, GE Healthcare), signals were recorded using the LAS 3000 Image Analyzer^® (Fujifilm, Tokyo, Japan) and quantified using the open source ImageJ software (developed by Wayne Rasband, NIH). ADAM10 migrates faster under non-reducing conditions than under reducing conditions. Therefore, detection by N-terminal ADAM10 antibody which requires non-reducing conditions, results in a 55 kDa band whereas the C-terminal ADAM10 antibody detects mature ADAM10 at 70 kDa under reducing conditions.

For analysis of cellular proteins cell lysates were generated using a lysis buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1% TritonX-100, 5 mM EDTA, 1 mM PMSF, 10 mM 1,10-phenanthroline monohydrate, one-fold Complete (Roche) and 10 µM GI254023X. After 20 min incubation on ice, lysates were cleared by 10 min centrifugation at 16,100× g and protein content was analysed via a BCA assay (Interchim, Montluçon, France) according to manufacturer’s instructions.
Samples from cell lysates (20 µg total protein), were heated in reducing SDS sample buffer (250 mM Tris HCl (pH 6.8), 50% (w/v) glycerol, 10% (w/v) SDS, 0.1% bromophenol blue, 5% β-mercaptoethanol) and subjected to SDS-polyacrylamide gel electrophoresis using 10% Tris-glycine gels. Proteins were transferred onto polyvinylidene difluoride membranes (Hybond-P, Amersham). Membranes were blocked with 5% (w/v) non-fat dry milk in TBS with 0.05% Tween for 1 h and probed with primary antibodies for 1 h at room temperature or overnight at 4 °C followed by incubation with HRP-coupled secondary antibodies (diluted 1:30,000) for 1 h. After addition of enhanced chemiluminescence substrate (ECL Prime, GE Healthcare), signals were recorded using the LAS 3000 Image Analyzer^® (Fujifilm, Tokyo, Japan).

Mouse cortices were homogenized in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.0% NP-40, and a protease inhibitor cocktail (Roche). Cell lysates were boiled in sample buffer for 5 min. The proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membrane was blocked with 5% nonfat dry milk in PBS containing 0.05% Tween-20 (PBS-T) and then incubated with primary antibody diluted in PBS-T containing 1% nonfat dry milk for 1 h at room temperature or overnight at 4°C. After washing in PBS-T, the membrane was incubated with horseradish peroxidase–conjugated secondary antibodies. An enhanced chemiluminescence system (GE Healthcare) was used for detection. Signals were detected and quantified using an LAS-3000 image analyzer (Fuji Film). Class III β-tubulin was used as a loading control.