FIGURE 3.

Expression of the methylation site L309Δ C subunit mutant affects the association of PP2A with the plasma membrane. Control and stable N2a cell lines expressing HA-tagged wild-type C (N2a-Wt C) or the L309Δ mutant of PP2A C subunit (N2a-L309Δ) were analyzed by Western blotting after subcellular fractionation. A, top panel, analysis of total homogenates (∼30 μg of proteins each) with anti-C antibody shows similar expression levels of ectopic proteins in N2a-Wt C and N2a-L309Δ cells. Lower panel, immunoblot analysis with anti-HA antibody of R and NR membrane fractions (∼20 μg of proteins each) purified from the same cells reveals that the L309Δ mutant is essentially excluded from rafts and present at low levels in non-rafts. B, quantitative analyses show that in contrast to the HA-tagged L309Δ mutant, the distribution of HA-tagged Wt C subunit in rafts is similar to that of endogenous C (Endo. C) (n = 4, mean ± S.D.; *, p < 0.001, relative to endogenous C). C, quantitative analyses show that relative to control cells, total raft- (black bars) and non-raft (gray bars)-associated PP2A C levels are increased in N2a-Wt C cells; in contrast, there is a significant loss of R- and NR-bound PP2A C subunit in N2a-L309Δ cells (n = 4, mean ± S.D.; *, p < 0.001, relative to controls). D, representative blot from three separate experiments showing the nuclear enrichment of demethylated C and PME-1 in N2a-L309Δ cells relative to N2a-Wt C cells. LSD-1, nuclear marker.