Fig. 6.
Cytochrome c restores PP2A enzymatic activity by sequestering its inhibitor ANP32B. (A) Left panel — Western-Blot against c-myc tag, encoded at the C-terminal of ANP32B1-251, after performing pull-down assays. Central panel — Western-Blot against the catalytic subunit of PP2A (PP2A-C), demonstrating that the endogenous enzyme was captured by means of the Ni-NTA sepharose matrix. Right panel — Western-Blot against c-myc tag of cell lysates as loading and transfection control. (B–D) Relative PP2A enzymatic activity in HEK293T cell extracts transfected with either pcDNA 3.1-ANP32B1-251-c-myc (B), pcDNA 3.1-ANP32B1-161-c-myc (C) or pcDNA 3.1-MBP-ANP32B162-251-c-myc (D), upon addition of increasing amounts of recombinant Cc. Cells transfected with empty pcDNA 3.1 were used as control (left bar in all panels). Each activity value corresponds to the average ± S.D. of three independent experiments. Detection by Western-Blot of transfected ANP32B species in cell extracts shown in panels B–D, by using anti-c-myc antibody, are included.