Isolation and Certification of Endoplasmic Reticulum–mitochondria Contacts (ERMC) from Mouse Lung
Tissues DONG Shu-an,YU Jian-bo,WU Jian-hua
Department of Anesthesiology, Tianjin Nankai Hospital,Tianjin (300100), China.
Abstract:
Objective To isolate and identify ERMC from lung tissue of C57BL/6 mice. Methods Ten8-10 weeks old C57BL/6 male mice were selected. After killing the mice, 1 g of lung tissue homogenate was taken. Endoplasmic reticulum, crude mitochondria, ERMC and purified mitochondria were separated by gradient centrifugation. The above structures were identified by Western blotting. Results Gradient centrifugation contained ERMC in a white band in the middle of the centrifugal tube. The bottom white sediment was purified mitochondria. Subsequently, we identified the purified ERMC and other components. The results showed that PDI was mainly expressed in endoplasmic reticulum and ERMC, Cyto C was mainly distributed in mitochondria,while calcineurin was mainly expressed in endoplasmic reticulum, IP3R-1 was the calcium dissociation in endoplasmic reticulum. Subchannels were expressed in endoplasmic reticulum and ERMC, and Sig-1R was mostly expressed in ERMC and mitochondria. Conclusion The results of the molecular expression distribution of the characteristic proteins mentioned above are consistentthe literature, indicating that our method of isolating and purifying ERMC in lung tissue is effective and can be used in future experiments.
