Hrp labeled goat anti rabbit igg antibody

The area of ischemia was defined as the region appearing pale. The corresponding areas of the heart were divided into different samples. The samples of each group (n=3) were homogenized, suspended in RIPA buffer (Sigma-Aldrich, Merck KGaA) and protease inhibitor cocktail (Roche Applied Science), and centrifuged at 14,000 × g for 10 min at 4°C. Protein concentration of the supernatant was measured by the bicinchoninic acid method; 50 µg of protein was separated by Tris-glycine 10% SDS-PAGE and transferred to a nitrocellulose membrane. Blocking was performed with 5% skimmed milk and 0.1% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. The membrane was incubated with rabbit anti-rat polyclonal GLP-1R primary antibody (1:500; cat. no. ab188602; Abcam) at 4°C overnight, washed with PBS, and incubated with HRP-labeled goat anti-rabbit IgG antibody (1:300; cat. no. ab6721; Abcam) at room temperature for 1 h. Chemiluminescence was performed with chemiluminescent HRP substrate using a SuperSignal West Pico Chemiluminescence Kit Detection System (Pierce, Thermo Fisher Scientific, Inc.). β-actin (1:500; cat. no. ab8227; Abcam) was used as internal control; each protein band was quantified using ImageJ software (v1.8.0, National Institutes of Health) and normalized to β-actin.

THP-1 human monocytic cell line was purchased from American Type Culture Collection (cat. no. TIB-202). Mouse anti-PPARγ antibody (product code ab41928), rabbit anti-tartrate-resistant acid phosphatase (TRAP) antibody (product code ab65854) and rabbit anti-HSP90α antibody (product code ab79849) were acquired from Abcam. Rabbit anti-GAPDH (cat. no. ab8245; Abcam) antibody, HRP-labeled goat anti-rabbit IgG antibody (cat. no. #7074S) and goat anti-mouse IgG antibody (cat. no. #7076S)were provided by Cell Signaling Technology, Inc. Alvespimycin was obtained from MedChemExpress (product no. HY-10389). High-glucose Dulbecco's modified Eagle medium (Dulbecco's modified Eagle medium, DMEM), fetal bovine serum (FBS) and other reagents for cell culture were provided by Gibco; Thermo Fisher Scientific, Inc. TRAP kits were products of Sigma-Aldrich; Merck KGaA (cat. no. 387A). Cell lysis buffer, protease inhibitors and other reagents were purchased from Beyotime Institute of Biotechnology. Chemiluminescence detection reagent pro-light HRP was purchased from MilliporeSigma.