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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Cell Signal. 2015 Feb 26;27(6):1186–1197. doi: 10.1016/j.cellsig.2015.02.024

Figure 5.

Figure 5

Accumulation of Ub-protein conjugates is associated with mitoROS-dependent activation of AMPK. Peritoneal macrophages were incubated with or without MG312 (10 µM) for 2 hours followed by staining cells for Ub-protein and GRP75, a mitochondrial marker. (A and B) Representative images show mitochondria (red), Ub-protein conjugates (green) and nuclei (blue). Area of interest (dotted lines in A) is magnified and shown in panel (B). Arrows indicate overlap between mitochondria and Ub-protein conjugates. (C) The amount of Ub-protein conjugates, VDAC, and α-tubulin was determined using Western blot analysis of whole cell or mitochondrial extracts obtained from Raw 264.7 that were treated with MG132 (0 or 10 µM) for 60 minutes. (D). Raw 264.7 cells were pretreated with PYR41 (0 or 50 µM) for 30 minutes followed by exposure to MG132 (0 or 10 µM) for additional 60 minutes. Ub-protein conjugates obtained from mitochondrial fractions are shown. (E) The extent of ROS production was determined in Raw 264.7 macrophages pre-treated with PYR41 (0 or 50 µM) for 30 minutes followed by inclusion of MG132 (0 or 10 µM) for additional 60 minutes. MitoSOX fluorescence intensity was determined using flow cytometry.