FIGURE 1.

CaN is the major Drp1 Ser-656 phosphatase in brain and heart tissue. A, GST-Drp1-(585–662) was phosphorylated in vitro with [γ-³²P]ATP and PKA and used as a substrate for phosphatases in digitonin extracts from adult rat brain (top) and heart (bottom). The activities of PP1, PP2A, PP2B (CaN), and PP2C were isolated by the addition of specific activators or inhibitors as indicated. Reactions were terminated after 3, 10, and 30 min by the addition of 10% TCA. Soluble ³²P was quantified by liquid scintillation counting and expressed as percent of input (mean ± S.E. from four independent experiments). B, Western blots of the above tissue extracts for the catalytic subunits of CaN (CNAα/β), PP1, and PP2A; ERK is a loading control.