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. 2021 Jan 7;296:100237. doi: 10.1074/jbc.RA120.016069

Figure 5.

Figure 5

PP2A methylation state affects APP membrane distribution and Fyn-dependent APP processing in N2a cells.A, representative immunoblots of endogenous APP distribution in purified membrane fractions from control or WT-, L309Δ-, LCMT1-, or PME1-expressing N2a cells. Quantitative analyses of the immunoblots from n = 4 separate experiments confirmed that there were no statistically significant changes in APP expression levels (p > 0.05) in total lysates from these cells. B, relative levels of membrane-associated APP in these cells. Data are mean ± SEM from n = 3 separate purifications and were analyzed using one-way ANOVA (F (4, 10) = 99.2, p < 0.0001) with post hoc Dunnett’s test. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, versus control. C, representative immunoblots of APP distribution in aliquots of the same purified N2a cell raft and nonraft fractions analyzed in Figure 2E. Levels of expressed HA-tagged proteins and APP in corresponding total cell lysates are shown for reference. D, relative levels of raft-associated APP were quantified in EV-, WT-, and L309Δ-expressing N2a cells and are expressed as the percent of total membrane-associated APP. Data (mean ± SEM from n = 3–5 separate purifications) were appraised using one-way ANOVA (F (2, 9) = 382.5, p < 0.0001) with post hoc Dunnett’s test. ∗∗p < 0.01, ∗∗∗∗p < 0.0001, versus control. E, levels of secreted sAPPα species and corresponding cellular APP levels were comparatively analyzed by Western blot in WT- and LCMT1-expressing N2a cells, relative to control N2a cells, after incubation for 4 h in a conditioned media in the absence or presence of 5-μM PP2 or PP3. F, the release of sAPPα was quantified in these cells after normalization for total cellular APP levels. Data shown are the mean ± SEM from 3 separate assays and were analyzed using two-way ANOVA (cell line: F = 73.57, p < 0.0001; treatment: F = 112.7, p < 0.0001; interaction: F = 6.46, p = 0.01) using Tukey’s post hoc multiple comparisons test. ##p < 0.01, ####p < 0.0001, versus control N2a; ∗∗∗∗p < 0.0001; ns, not significant. APP, amyloid precursor protein; HA, hemagglutinin; LCMT1, leucine carboxyl methyltransferase 1; N2a, Neuro-2a; PP2Ac, catalytic “C” subunit of PP2A.