Ubqln1

Hypothalamus isolated from the brain and the gastrocnemius muscle were collected from Wt and Tg mice for Western blot analysis at 18 months of age. In addition, stable MEFs derived from Wt and Tg mice were harvested for Western blot analysis. Immunoblotting was conducted as previously described (Lü and Wang 2012). Primary antibodies consisted of Ubqln1 (1:1000, Abcam, Cambridge, MA), SIRT1 (sirtuin1) (1:250 Cell Signaling, Danvers, MA), AMPKα (AMP‐dependent protein kinase‐α) (1:1000, Cell Signaling), GAPDH (1:1000, Cell Signaling) and Actin (1:1000, Santa Cruz). Appropriate secondary antibodies used were conjugated with fluorescent dyes (LI‐COR Inc., Lincoln, NE). The bands were detected using an Odyssey scanner (LI‐COR) and quantified using UN‐SCAN‐IT gel6.1 software (Silk Scientific Inc., Orem, Utah).

Cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche) for 30 min and then centrifuged to remove the Triton-insoluble pellet. Samples normalized with equal protein concentration were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. Polyvinylidene fluoride (PVDF) membranes were incubated with the following primary antibodies for overnight at 4°C: UBQLN1 (Abcam, 1:2000), p-mTOR (Abcam, 1:2000), mTOR (Abcam, 1:2000), p-p70S6K (Abcam, 1:2000), p70S6K (Abcam, 1:2000), GAPDH (Sigma, 1:1000). Goat anti-mouse (1:5000) or anti-rabbit (1:5000) secondary antibodies (BBI Life Science Corp.) were used. The protein bands were visualized using chemiluminescence imaging system V1.8.0.112 (Tannon Science & Technology Co., Ltd,). All experiments were independently performed in triplicates [19 ].