Trans blot turbo mini pvdf transfer pack

The Ucp1 protein content in BAT was determined by Western blot. Tissues were homogenized in RIPA (radio immunoprecipitation assay lysis buffer), and the protein was extracted and stored at − 20 °C. The protein content was quantified using a BCA protein assay kit (Pierce, Rockford, IL, USA) following manufacturer’s instructions.
First, 15 µg of protein in Laemmli loading buffer was denatured, loaded onto 10% acrylamide gels made with TGX™ Fast Cast™ Acrylamide Solutions (Bio-Rad, Barcelona, Spain) and run at 90 V for 75 min. Gels were then transferred onto a PVDF membrane using the Trans-Blot Transfer System (Bio-Rad, Barcelona, Spain) with Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad, Barcelona, Spain) following the manufacturer’s instructions. The membrane was blocked and then incubated with anti-Ucp1 antibody (Abcam, Cambridge, United Kingdom) at 4 °C overnight. Afterwards, the membrane was incubated for 2 h with the secondary antibody (GE Health Care Life Sciences, Barcelona), and the protein was detected with the chemiluminescent reagent ECL Select Western Blotting Detection Reagent (GE Healthcare, Barcelona, Spain). Protein levels were quantified with the open source software ImageJ [22 (link)] and normalized to β-actin protein levels.

To quantify HC1 and HC2 protein expression, first instar larvae dissected from the uteri were homogenized twice in ice-cold lysis buffer which contained 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% Na-deoxycholate, 0.5% NP-40, 0.5% SDS, and protease inhibitor (EDTA-free). Lysates were cleared by centrifugation at 15.000×g at 4 °C for 30 min, and the protein content was determined by a protein assay using bovine serum albumin (BSA) as a standard (Bradford method). Sixty micrograms of protein from each sample was reconstituted directly in the appropriate amount of sample buffer and separated in Mini-Protean TGX System Precast Protein Gels (Bio-Rad, Hercules, CA, USA) and transferred to Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad, Hercules, CA, USA). The membranes were washed and blocked in 0.02 M Tris-buffered saline containing 5% BSA and 0.1% Tween 20 and then incubated overnight at 4 °C with anti-HC1 or HC2 antibodies diluted 1:500. Next, the membranes were washed with TBST (Tris-buffered saline containing 0.1% Tween 20) and incubated for 1 h with a horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz, USA) diluted 1:1000. Signals were detected by chemiluminescence using WesternBright Quantum HRP substrate (Advansta Inc., Menlo Park, USA) and visualized using the Chemidoc™ XRS + System (Bio-Rad, Hercules, USA).

Cells were lysed and loaded onto 10% precast polyacrylamide gels (Mini-PROTEAN TGX Precast Gels; Bio-Rad, Hercules, CA, USA); they were then blotted onto polyvinylidene difluoride (PVDF) membranes (Trans-Blot Turbo Mini PVDF Transfer Packs; Bio-Rad). After the membranes had been blocked blocking Tween 20 in PBS containing 5% non-fat dry milk, they were incubated with primary antibodies against p-PI3K (abs130868; Absin Bioscience, Inc., Shanghai, China), p-AKT (4060S; CST, Danvers, MA, USA), FOXO (2880T; CST), and Cyclin D1 (AF1183; Beyotime) at 4°C overnight. Membranes were incubated with a horseradish peroxidase–onjugated secondary anti-rabbit (WB0177) and IgG (WB0176), as appropriate. Relative expression was determined using Image-Pro Plus software (v. 6.0; Media Cybernetics, Rockville, MD, USA).

HAECs were starved overnight in endothelial cell basal medium (EBM-2; PromoCell GmbH). After treatment with reagents for 1 h, cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, and 0.1% SDS) containing protease and phosphatase inhibitors (100× Halt Protease and Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific Inc., Waltham, MA, USA). Protein concentrations were measured using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA, USA). Cell lysates (25 μg/lane) were subjected to 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Trans-Blot Turbo Mini PVDF Transfer Packs, Bio-Rad). After blocking with 5% skim milk in Tris-buffered saline, the membranes were incubated overnight at 4°C with primary antibodies against PDE3A, PDE3B, Epac-1, PKA RIIβ (1:200 dilution), phospho-PDK1, PDK-1, phospho-Akt, Akt, phospho-MAPK, MAPK, Rap-1A/B (1:250), phospho-PLA₂, or PLA₂ (1:100), followed by incubation with secondary antibodies (1:1000 dilution). The membranes were developed using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.) followed by exposure to a CCD camera (Luminescent image analyzer, GE Healthcare UK Ltd., Buckinghamshire, UK), and analyzed using Image quant LAS4000 software (GE Healthcare UK Ltd.).