Isotype igg

Wild-type male mice (C57BL/6J) were obtained from Beijing Vital River Laboratory Animal Technology and maintained in a temperature-controlled animal vivarium with adequate food and water. Antiresistin neutralizing antibody (nAb) is purchased from Merck Millipore. Isotype IgG is purchased from Thermo Fisher Scientific. First, 48 mice were divided into 2 groups: the DOX and the saline group; the DOX group was administered 15 mg/kg DOX by a single intraperitoneal injection, and the other group was administered saline as a control (N = 24). Then, every other day, we harvested 4 mice from each group to obtain cardiac and serum resistin levels. In addition, another 40 mice were divided into 4 groups: saline-IgG, saline-nAb, DOX-IgG, and DOX-nAb (N = 10). The saline-nAb group and the DOX-nAb group were pretreated with 200 μg of mouse antiresistin nAb while the same dose of Isotype IgG for the saline-IgG and DOX-IgG groups. After one hour, the mice were treated with 15 mg/kg DOX or saline by a single intraperitoneal injection (N = 10). All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Ningbo First Hospital (Approval No. 2021DC2408).

Proteins were extracted using a non-denaturing buffer containing Tris-HCl (pH8, 20 mM), NaCl (137 mM), NP40 (1%), EDTA (2 mM) and supplemented with protease inhibitors. Following extraction, proteins were incubated under rotation at 4 °C for 30 min and centrifuged at 14,000 g for 15 min at 4 °C to eliminate cell debris. 5 µg antibodies were incubated overnight with 500 µg of the protein extract (except for the IP performed from MAMs extracts, where 250 µg were incubated). We used isotype IgG as a control (Thermo Scientific, Waltham, MA). Then, protein A/G magnetic beads (Thermo Scientific, Waltham, MA) were added and incubated at 4 °C under rotation for 2 h. After three washes with a low salt buffer containing SDS (0.1%), Triton X-100 (1%), EDTA (2 mM), Tris-HCl pH 8 (20 mM) and NaCl (150 mM) and one wash of high salt buffer composed of SDS (0.1%), Triton X-100 (1%), EDTA (2 mM), Tris-HCl pH 8 (20 mM) and NaCl (450 mM), proteins were eluted at 99 °C from magnetic beads using Laemmli’s buffer and then processed for western blotting.