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. Author manuscript; available in PMC: 2014 Jun 1.
Published in final edited form as: Nat Cell Biol. 2013 Nov 10;15(12):1464–1472. doi: 10.1038/ncb2868

Figure 2.

Figure 2

MCU regulates mitochondrial calcium uptake in permeabilized MEFs. a) Comparison of cytosolic calcium levels in permeabilized WT and MCU-/- MEFs. Arrows indicate calcium addition. Increasing cytosolic calcium results in a rapid increase in the fluorescent signal in both cell types but the subsequent decline in the fluorescent signal, representing mitochondrial calcium uptake, is only observed in WT cells. Shown is one tracing that is representative of three similar experiments. b) Ruthenium red (Ru360; 3 μM) inhibits mitochondrial calcium uptake in permeabilized WT MEFs. c) Western blot analysis of MCU expression. MCU-/- MEFs were infected with retroviruses encoding an epitope-tagged form of either WT MCU or MCUmut containing two amino acid substitutions (at amino acid 261 and 264) known to abrogate uniporter activity. The tagged constructs migrate slightly slower than endogenous MCU but were expressed at roughly endogenous levels. d) Cytosolic calcium measurements in permeabilized WT MEFs, MCU-/- MEFs, MCU-/- MEFs reconstituted with wild type MCU and MCU-/- MEFs reconstituted with MCUmut.