1. Tissue homogenate with precipitation Tissue homogenate without precipitation Cy5: PP2A 0.1 U Cy5: PP2A 1U Cy3: PP2A 0.1 U Cy3: PP2A 1U Cy5: PP2A 1U Supplementary Figure 2 pH 7pH 4

2. Cy5: PP1 1U Cy3: PP1 1UCy3: PP1 10U Cy5: PP1 10U Cy5: PP1 1U Cy3: PP1 1U Tissue homogenate with precipitation Tissue homogenate without precipitation Supplementary Figure 3 pH 7pH 4

3. Cy5: AP 1U Cy3: AP 1U Cy3: AP 10U Cy5: AP 10U Cy3: AP 10U Cy5: AP 10U Tissue homogenate with precipitation Tissue homogenate without precipitation Supplementary Figure 4 pH 7pH 4

4. Cy5: -PP 10U Cy3: -PP 10U Cy3: -PP 100U Cy5: -PP 100U Cy3: -PP 100U Tissue homogenate with precipitation Tissue homogenate without precipitation Supplementary Figure 5 pH 7pH 4

5. Cy5: AP 10U, pH 9 Cy3: AP 10U, pH 9 Cy5: AP 10U, pH 9 Tissue homogenate with precipitation Tissue homogenate without precipitation Supplementary Figure 6 pH 7pH 4

6. Mitochondrial-enriched fraction resuspended in SDS/HEPES buffer (H-buffer) Mitochondrial-enriched fraction resuspended in phosphatase buffer (P-buffer) Cy3: AP 10U Cy5: -PP 100U Cy5: AP 10U Cy3: -PP 100U Cy5: AP 10U Supplementary Figure 7 pH 7pH 4

7. 0.5U AP:Cy3 (green) 0.5U PP1: Cy5 (red) 0.5U PP2A: Cy2 (blue) Supplementary Figure 8 pH 7pH 4