ARPP19 is functional in human cells. (A) Synchronized HeLa cells were lysed in the presence of a reversible cross-linker and used for immunoprecipitation (IP), using anti-NterARPP19 or anti-GST (control [CT]) antibodies. PP2A-B55 subunits and ARPP19 were checked in inputs and immunoprecipitates (IP). The levels of PP2A subunits A and C in the IPs were quantified using ImageJ software, normalized to the total levels of ARPP19, and represented. Values (mean ± SD) are from two different experiments. (B) HeLa cells were synchronized in G1 (24-h thymidine block), early S phase (2.5-h thymidine release), late S phase (6-h thymidine release), G2 (12-h RO3306 block), or M phase (12-h nocodazole shake-off) and lysed in the presence of a reversible cross-linker and microcystin. ARPP19 IPs and whole-cell extracts were used for Western blotting. (C) Nocodazole-blocked HeLa cells were released, lysed in the presence of microcystin, and used for IP. Inputs and IP were analyzed by Western blotting with the indicated antibodies. (D) Mitotic-enriched cell populations were lysed in the presence of a reversible cross-linker, and then microcystin was added (or not) at different time points, as indicated in the scheme, before IP using anti-NterARPP19 or anti-GST (CT) antibodies. Inputs and IPs were then used for Western blotting. The phosphorylated ARPP19 at S62/total ARPP19 ratio intensities at different time points after cell lysis of this experiment are shown in the lower graph. Data are representative of two different experiments.