Figure 6. Analysis of the accumulation of the high-molecular-mass Ppp5 and immunocytological localization of endogenous Ppp5 after treatment with nocodazole.

(A) Immunoblot analysis of MCF7 cells. Con, untreated control MCF7 cells; NA, adherent cells after 500 nM nocodazole for 18 h; NR, detached cells from nocodazole treatment; Rec, adherent cells 24 h after removal of nocodazole. After the above treatments, cells were collected in NuPAGE sample loading buffer and incubated at 70 °C for 15–30 min. Electrophoresis was in Mops buffer, and blots were prepared and probed with antibodies as indicated in the Ppp5 antibody map (Figure 5A), except that the S353 antibody is not shown, because it recognizes the C-terminus only weakly and gave several artifactual bands with lysates. (B) Indirect immunofluorescence of endogenous Ppp5 in MCF7 cells treated as in (A). Ppp5 was visualized using S731, S412 and S411 antibodies, and anti-sheep IgG-FITC secondary antibody with an inverted microscope connected to Openlab digital imaging software (Improvision). Phase-contrast images of the same cells are shown below the immunofluorescent panels. (C) Comparison of the detection of endogenous Ppp5, p50 and p130 by different antibodies. Two lysates from HEK-293 cells treated with nocodazole were immunoblotted with antibodies against the N-terminus (S731), TPR domain (S412) and phosphatase domain of Ppp5 (S411). Note the different intensities of Ppp5, p50 and p130 with the different antibodies. (D) DAPI (4,6-diamidino-2-phenylindole) staining of the detached cells merged with the signal from anti-TPR antibodies (left-hand panels); control with anti-sheep secondary antibody alone and the phase-contrast image of the same cells (right-hand panels).