Insulin Signaling in Tendon Cells

Primary tenocytes were isolated from C57Bl/6 J mice and used up to passage 3. Whole FTs were isolated from HFD and LFD-fed mice 48 weeks after diet initiation (n = 3 per group). Tenocytes and tendons were stimulated with either vehicle (0.5% BSA in PBS) or 1 nM insulin for 15 minutes, followed by protein isolation for western blot analyses. Blots were probed for phospho-AKT Ser473 (1:1000, Cell Signaling, #4060), and total AKT (1:1000, Cell Signaling, #9272 S)^{33 (link)} and were developed with SuperSignal West Pico or Femto Chemiluminescent Substrate and imaged on a GelDocXR (BioRad, Hercules, CA). To assess changes in IRβ expression in IRcKO^S100a4 mice, total protein was extracted from WT and IRcKO^S100a4 tendons (n = 3 per genotype) for western blot analyses. Blots were probed with IRβ (1:300, Santa Cruz, #sc-20739)^{34 (link)} and β-actin (1:2500, Sigma Aldrich, #A2228)^{35 (link)}, and were developed with SuperSignal West Pico or Femto Chemiluminescent Substrate and imaged on a GelDocXR (BioRad, Hercules, CA).