Image lab software 5

Fibroblast cells grown on a 100‐mm dish were harvested with 0.05% trypsin‐EDTA when they reached 70% confluence and rinsed with ice‐cold PBS twice. Nuclei were separated from cytoplasm following the manual of NE‐PER Nuclear and Cytoplasmic Extraction Reagents (#78835; Thermo Scientific). After centrifuging, the cytoplasm supernatant was removed. The pellets containing nuclei were resuspended well in lysis buffer (50 mm Tris–HCl, pH 7.4, 150 mm NaCl, 1% Triton X‐100, 0.1% deoxycholate, complete mini protease inhibitor cocktail tablet) and subjected to slight sonication at 20% amplitude for 30 s. (FB120; Fisher Scientific). The whole nuclei lysate was further centrifuged at 16 000 g for 5 min at cold. The supernatant was saved as the soluble fraction of the nuclei while the pellet was saved as the insoluble fraction of the nuclei. Both fractions of nuclei were prepared for Western blot assay by adding Laemmli sample buffer (Bio‐Rad). A one‐fifth portion of either soluble or insoluble fraction sample was loaded onto 10% SDS–PAGE gel and then proceeded for Western blot analysis. Images were taken with ChemiDoc^™ Touch Imaging System (Bio‐Rad), and band intensity analysis was carried out with Image Lab software 5.2.1 (Bio‐Rad).

Mouse islets were preincubated for 1 h in 1 mmol/L glucose Krebs‐Ringer Bicarbonate (KRB) and subsequently treated for 20 min with P2Y₁ agonists, ATP (10 μmol/L) and MRS2365 (100 nmol/L; EC₅₀ 0.4 nmol), or 30‐mmol KCl ± P2Y₁ antagonist, MRS2500 (1 μmol/L; EC₅₀ 0.78 nmol). Following treatment, cells were lysed in buffer containing (in mmol/L): 20 Tris‐HCl (pH = 7.5); 150 NaCl, 1 EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 EDTA, 1 b‐glycerophosphate, 25 N‐ethylmaleimide, 1% Triton X‐100, and 1X protease inhibitor cocktail. Protein lysates were separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), transferred to nitrocellulose membranes, incubated overnight at 4°C with primary antibodies (phospho‐PKD/PKCmu (serine 916) Rabbit A [Cell signalling] and anti‐β‐actin; sc‐4778 [Santa Cruz Biotechnology]), and visualized with horseradish peroxidase‐labeled anti‐rabbit IgG as secondary antibodies (Amersham, Baie d’Urfe, PQ). Images were acquired using a ChemiDoc MP System (Bio‐Rad) and analyzed using the densitometry analysis function in Image Lab Software 5.2.1 (Bio‐Rad) and normalized to actin as a loading control and expressed as a fold increase from baseline.