Pkf115 584

BrdU, tamoxifen and clodronate disodium were purchased from Sigma. Human complement C1q and C1 complexes were from Calbiochem. AngII and hydralazine hydrochloride were from Wako. C1-INH (Berinert) was from CSL Behring. PKF115-584 (ref. 22 (link)) was from Novartis. Human recombinant Wnt3A was from R&D. Mouse recombinant IL-4 was from PeproTech. Mouse monoclonal antibody (14/Beta-Catenin) against β-catenin was from BD Transduction Laboratories (immunofluorescence (IF) dilution 1:200). Rat monoclonal antibody (clone CI:A3-1) against mouse F4/80 (IF dilution 1:50), rabbit monoclonal antibody (clone E247) against β-catenin (WB dilution 1:2,000), rabbit polyclonal antibody against axin2 (WB dilution 1:2,000, IF dilution 1:100) and rat monoclonal antibody against BrdU (clone BU1 75(ICR1)) (IF dilution 1:200) were from Abcam. Mouse monoclonal antibody (clone 8E7) against ABC was from Millipore (WB dilution 1:1,000). rabbit polyclonal antibody against actin and alpha-smooth muscle actin (αSMA) were from Sigma (IF dilution 1:200). TACS 2TdT Fluorescein Kit was from Trevigen. Mouse monoclonal antibodies against C1r (WB dilution 1:250) and C1s (WB dilution 1:250) were from R&D. Secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 546 were from Molecular Probes (IF dilution 1:200).

Men1^flox/flox mice (129/SvJ)13 (link) were bred with mice expressing Cre recombinase driven by the rat insulin promoter (Rip-Cre) to generate the βMen1-KO mice. Ctnnb1^flox/flox mice (C57BL/6J) were purchased from Jackson Laboratory. The βBcat-KO mice were generated by breeding Ctnnb1^flox/flox mice and Rip-Cre mice (mixed C57BL/6J:129/SvJ). Heterozygous (Men1^f/+-Cre⁺, Ctnnb1^f/+-Cre⁺) mice (mixed C57BL/6J:129/SvJ) were backcrossed with C57BL/6J mice five times and then crossed to generate homozygous mice. The βMen1/Bcat-KO mice (C57BL/6J) were generated by breeding βMen1-KO mice (C57BL/6J) and βBcat-KO mice (C57BL/6J). The C57BL/6J mice were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences (SLAC, CAS). All of the mice were housed in pathogen-free facilities with a 12 h light/dark cycle and had free access to water and food. The βBcat-KO and control mice were fed a standard chow diet or a HFD (research diets) for 4 months. βMen1-KO mice, at the age of 14 months, were intraperitoneally injected with PKF115-584 (0.5 mg kg⁻¹, Novartis Pharmaceuticals) or vehicle (0.2% dimethylsulphoxide in PBS) every 3 days for 8 weeks. All mice used for the experiments were males. All of the animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.