Irisin

Experimental procedures conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health; all animal procedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee (protocol AN-625, AN-3965). To generate mCaROCK1 mice, we bred MCK-Cre transgenic mice (Jackson laboratory, Bar Harbor, ME) with mice bearing the floxed, transcriptional stop cassette that is upstream of the constitutively active ROCK1 (Supplemental Fig. 1a)18 (link)24 (link). Mouse breeding strategy is shown in supplemental Fig. 1b. Muscle-specific ROCK1 activation was confirmed by ROCK activity assay (see below). For Irisin treatment experiment, mCaROCK1 and control mice were given Irisin (100 μg/Kg/day, ip) for 3 weeks. Irisin was purchased from (Cayman Chemical, Ann Arbor, MI). Mice were housed with a 12-h light-dark cycles and were given free access to food and water.

DMEM medium, antibiotics (streptomycin and penicillin), fetal bovine serum (FBS) were purchased from PAA (Pasching, Austria). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Irisin was purchased from Cayman. Annexin V kit, Fc block antibodies, Cytofix/Cytoperm solution, PerCP-Cy5.5 streptavidin, Cytometric Bead Arrays for JNK, ERK and p38 quantification were acquired from BD Biosciences Pharmingen (San Diego, CA, USA). Phospho-NF-κB (ser536) Alexa Fluor 647 Conjugate rabbit monoclonal antibody was acquired from Cell Signaling Technology (Beverly, MA, USA). Elisa kit for HMGB1 and IL-1β were purchased from IBL International (Hamburg, Germany). RNeasy Plus Mini Kit (74134) for RNA isolation was obtained from Qiagen (Hilden, Germany). High Capacity RNA-to-cDNA Kit (4387406) for reverse transcription and TaqMan Gene Expresson Master Mix (4369026) for real time PCR were obtained from Applied Biosystems (ThermoFisher; Foster City, CA, USA).