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After clearing cell debris by centrifugation at 4 degree, the extract was subjected to proteasomal activity assay using the Proteasome-Glo™ Chymotrypsin-Like Assay (Promega).
These data suggest that, in addition to the resident macrophages, additional cell types can be recruited to a site of acute cell death and participate in clearing cell corpses.
After clearing cell lysates by centrifugation (S1), we performed a high-speed centrifugation at 166,000 g and found that the large majority of ubiquitinated proteins that accumulated during the MG132 treatment were not soluble (P2; Fig. 2A).
The lysosome-associated CD68 protein is typically only expressed at the cell surface of phagocytically active cells, suggesting that this minor population may be actively clearing cell carcasses that are a consequence of normal thymopoiesis.
Hence, while M1 macrophages contribute to creating an inflammatory environment that helps clearing cell debris and activating satellite cells, M2 macrophages reduce inflammation and promote myogenic differentiation.
The data also argue that the snake venom enzyme is not only capable of clearing cell corpses but can aid in the demise of tumor cells that have initiated but not yet completed the death process.
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