Mouse tissue extraction
Reagents:
- Protease inhibitor cocktail from Roche, #11697498001.
This method is used to make extracts from mouse tissues except adipose tissues. The extracts are for regular SDS-PAGE and WB only. To measure phosphorylation, you need phosphatase inhibitors.
1. Harvest mouse tissues and snap freeze in liquid nitrogen.
2. Add 1 ml TNET (Tris, 50 mM, NaCl, 150 mM, EDTA, 1 mM, Triton X-100, 1%, pH 8.0) with protease inhibitors and homogenize until clear. Keep on ice.
3. Spin down at 14K RPM for 15 min at 4C.
4. Transfer the supernatant for further experiments.
Mouse adipose tissue extraction
1. Harvest adipose tissues and snap freeze in liquid nitrogen.
2. Add 1 ml TNE (No Triton X-100) with protease inhibitor and homogenize until clear. Keep on ice.
3. Spin down at 5000 RPM for 5 min.
4. Carefully remove the fat cake and resuspend the loose pellet. Alternatively, use the pipette tip to penetrate the fat cake and transfer the solution and the pellet to a new tube.
5. Add 10% Triton X-100 to a final concentration of 1-2%.
6. Spin down at 14K RPM for 15 min at 4C.
7. Transfer the supernatant for further experiments.
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Autophagy hits heart
Autophagy is acutely upregulated to provide necessary nutrients during starvation for survival. Under normal condition, constitutive autophagy is critical to perform house-keeping functions to eliminate damaged organelles and dysfunctional long-lived proteins.
Ageing is associated with accumulation of dysfunctional proteins in cells. Autophagic activity is decreasing along with ageing. Studies in worms and flies show that reconstitution of autophagy can increase life span. A recent report by Taneike and colleagues explored the role of autophagy in age-related cardiomyopathy.
In heart, autophagic activity is decreasing when ageing. Taneike et al eliminated autophagy in heart by knocking out an essential gene, ATG5, specifically in heart. The researchers found deteriorated cardiac function at 10 months of age in the deficient animals compared to wild type controls. The dysfunction of autophagy causes accumulation of dysfunctional mitochondrial, reduced mitochondrial efficiency and significant oxidative damage in cardiomyocytes. More interestingly, evidence of mitochondrial damage is obvious as early as 3 months old, before cardiac remodeling and dysfunction manifest. This study points to a possibility that autophagy is critical in maintaining cardiomyocyte homeostasis and autophagy may be a target for future therapeutic design for heart failure....
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