Figure 5. Subcellular localization of MKP1 and phosphorylated ERK after CPT treatment. (A) HCT116 cells were incubated for 24 h with CPT (500 nM), then cytoplasmic and nuclear fractions were collected and analyzed by western blotting using anti-MKP1 (MKP1), anti-phospho-ERKs (P-ERK), anti-ERKs (ERK), and anti-U1–70 antibodies. (B) HCT116 cells were incubated in the absence or presence of 500 nM CPT. After 24 h, cells were fixed with 4% (v/v) paraformaldehyde and immunostained for MKP1 using a FITC-conjugated secondary antibody and counterstained with PI. (a and d) Staining for PI (red). (b and e) Localization of MKP1 (green). (c and f) Merged images of MKP1 and PI. (C) Cells were stained as described in (B) except that the antibody against MKP1 was replaced with an antibody against phosphorylated ERKs. (a and d) Staining for PI (red). (b and e) Localization of phosphorylated ERKs (green). (c and f) Merged images of phosphorylated ERKs and PI.