Trim32 KO mice were previously described (22). TRIM32 KO and WT mice were maintained and treated according to approved protocols and in accordance with the institutional and national guidelines and regulations (IP00001489) approved by the Oregon Health and Science University. To analyze the autophagy response to dexamethasone, 3-month-old mice (n = 4 for each genotype) were injected subcutaneously with dexamethasone sodium salt (5 mg/kg; Sigma-Aldrich, D-1756) and prepared in a saline solution (vehicle, 0.9% NaCl). The control group received an injection of saline solution (vehicle, 0.9% NaCl). Eight hours after injection, mice were sacrificed, and quadricep muscles were harvested and deep-frozen in liquid nitrogen before storing at −80°C for subsequent protein extraction. For immunoblotting analysis, frozen muscles were crushed into a fine powder with a hammer, resuspended in the extraction buffer (Coimmunoprecipitation Kit, Thermo Fisher Scientific, 14321D), supplemented with protease, phosphatase, and deubiquitinase inhibitors as described above, homogenized using a Dounce homogenizer, incubated at 4°C for 30 min, and centrifuged at 13,000 rpm for 10 min to remove debris. Same amounts of total protein (30 μg per well) were loaded on SDS–polyacrylamide gel electrophoresis (PAGE) for immunoblotting analysis.

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