Goat anti mouse irdye 800cw secondary antibodies

Detection of UCP2 was performed by western blotting. Briefly, organs were perfused with 20 mM ethylenediaminetetraacetic acid (EDTA, pH 7.4). Liver tissues cut into small pieces were homogenized at 4°C in lysis buffer containing protease inhibitors (Roche, AG, Basel, Switzerland). Periepididymal adipose tissues were homogenized at 4°C in RIPA buffer with protease inhibitors (Roche, AG, Basel, Switzerland) and phosphatase inhibitor cocktails (Roche). Tissues were stored at -20°C for further protein quantification. Western blot analyses were performed with whole liver and adipose tissues lysates (40 μg of proteins) using anti-UCP2 (1:1000 dilution, Abcam) and anti-β-actin (1:15000 dilution, Sigma). Detection was performed with the Super Signal Chemiluminescence kit (Pierce), exposing the membrane to an autoradiograph film (GE Healthcare). Bands were digitalized and analyzed by size and intensity by the Image Master 2D Elite program. Anti-AMPK (Cell Signaling) was used, and infrared-labeled goat anti-mouse IRDye 800CW secondary antibodies (Li-Cor Biosciences) were added to bind to the primary antibody. Detection was performed with an Odyssey scanner. Bands were digitalized and analyzed by size and intensity by the Image Studio 3.1 [30 (link)].

RPE cells were cultured for 14 days and lysed by RIPA buffer (Beyotime, Shanghai, China) for 30 min. After centrifugation, total protein concentrations of all samples were quantified using a BCA Protein Quantification Kit (Vazyme) according to the manufacturer’s protocols. Equal amounts (30 μg) of proteins per lane were loaded and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel, and then transferred to nitrocellulose membranes. After blocking with PBS containing 3% BSA for 1 h at room temperature, the membranes were incubated with primary antibodies, including rabbit anti-PRPF6 (1:1000, Invitrogen), rabbit anti-CRALBP (1:1000, Proteintech), mouse anti-RPE65 (1:1000, Abcam), rabbit anti-tyrosinase (1:1000, Abcam), mouse/rabbit anti-GAPDH (1:2000, Arigo) and mouse anti-β-Actin (1:2000, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Then, membranes were incubated with goat anti-rabbit IRDye 680RD secondary antibodies or goat anti-mouse IRDye 800 CW secondary antibodies (1:10,000, LI-COR Bioscience, Lincoln, NE, USA) for 1 h at room temperature. The bands were imaged on the Odyssey Fc Imaging System (LI-COR Bioscience), and the results were analyzed by Fiji/Image J software (National Institutes of Health, Bethesda, MD, USA).

Megakaryocytes were lysed in 1 × RIPA lysis buffer (Beijing Solarbio Science & Technology, cat No.R0010, Beijing, China) supplemented with a fresh protease and phosphatase inhibitor cocktail. The protein concentration was determined using a protein assay kit (Beyotime Biotechnology, cat No.P0010, Haimen, China). SDS PAGE electrophoresis was used to separate total proteins and nitrocellulose membranes were applied for protein transfer. After being blocked with 5% non-fat milk, membranes were incubated with specific primary antibodies at 4 °C overnight, followed by 1 h of further incubation with goat anti-rabbit IRDye 800CW or goat anti-mouse IRDye 800CW secondary antibodies (LI-COR Biosciences, Lincoln, NE) at room temperature. The membranes were visualized with Infrared Imagine System (LI-COR Biosciences, Lincoln, NE). The primary antibodies used in this study were rabbit-anti-P-Myosin light chain (1:500, Cell Signaling, cat No.3671, USA), and mouse-anti-mouse β-actin (1:1000, Beyotime, cat No.AA128, Haimen, China). The densitometry measurements of scanned blots were assessed using Image J software (NIH, Maryland, USA).