Ecl advance

Cells in six-well plates were washed twice with ice-cold PBS and placed immediately in lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail I (Calbiochem), and phosphatse inhibitor cocktail V (Merk). Lysates were gently mixed for 10 min at 4°C and then centrifuged at 13,000×g for 15 min at 4°C. The protein concentration of the extracts was determined according to the method of Bradford, using BSA as the standard. Samples were separated by SDS-PAGE and transferred to PVDF-Plus membranes (Bio-Rad, Hercules, CA). The transferred membranes were blocked, washed, and incubated with various primary antibodies, followed by horseradish peroxidase-conjugated secondary antibody. Blotted membrane was developed with ECL Advance (Cell Signaling Technology, Boston, MA) and imaged with a LAS-4000 Super CCD Remote Control Science Imaging System (Fuji, JAP).

The cultured islets in the 6-well plates were washed twice with ice-cold phosphate-buffered saline (PBS) and immediately placed into a lysis buffer containing 25 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.4), 1% Nonidet P-40, 100 mmol/l NaCl, 2% glycerol, 5 mmol/l NaF, 1 mmol/l ethylenediamine tetraacetic acid (EDTA), 1 mmol/l Na₃VO₄, 1 mmol/l sodium pyrophosphate (NaPPi), 1 mmol/l phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 5 μg/ml leupeptin and 5 μg/ml pepstatin. Lysates were centrifuged at 14,000 × g for 10 min at 4°C. The protein concentration of the extracts was determined according to the Bradford method, using BSA as the standard. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 8% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF)-Plus membranes (Bio-Rad, Hercules, CA, USA). The transferred membranes were incubated with anti-Hmgcr antibody at 1:1,000 dilution overnight at 4°C. Primary antibodies were detected with donkey anti-rabbit IgG conjugated with horseradish peroxidase at 1:2,000 for 1 h at room temperature. The blotted membrane was developed with enhanced chemiluminescence (ECL) Advance (Cell Signaling Technology, Inc., Boston, MA, USA) and imaged with a LAS-4000 Super CCD Remote Control Science Imaging system (Fujifilm, Tokyo, Japan).