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. 2021 Nov 10;41(45):9431–9451. doi: 10.1523/JNEUROSCI.1914-20.2021

Figure 9.

Figure 9.

Tau-induced increases in active PP1 is dependent on PAD. PAD, a conformationally displayed biologically active motif in the extreme amino terminus of tau, was deleted in WT and P301L tau by removing amino acids 2–18 (Δ2–18). A, B, The full-length and Δ2–18 constructs were expressed in HEK293 cells, in addition to PP1α, at similar levels (B; WT tau, 4.02 ± 0.54; Δ2–18 WT, 3.24 ± 0.63; P301L, 5.32 ± 1.52; Δ2–18 P301L, 4.04 ± 0.58). C, Levels of active PP1α (as indicated by changes in inactive pT320 PP1) were identified using a phospho-Thr320 PP1 antibody (red), and total levels were detected using an anti-HaloTag antibody (green). D, Deletion of PAD resulted in significantly lower levels of active PP1 compared with expression of full-length tau in both WT and P301L backgrounds (WT tau, 0.3409 ± 0.0366; Δ2–18 WT, 0.4232 ± 0.0336; P301L, 0.2147 ± 0.0507; Δ2–18 P301L, 0.4062 ± 0.0406). E, F, WT tau, Δ2–18 WT, P301L, and Δ2–18 P301L were expressed at similar levels (F) along with PP1β (WT tau, 1.71 ± 0.14; Δ2–18 WT, 1.64 ± 0.28; P301L, 1.68 ± 0.24; Δ2–18 P301L, 1.78 ± 0.41). G, Immunoblotting of total PP1 (green) and inactive PP1β (red, pT320 PP1 antibody corresponds to pT316 in PP1β). H, Active PP1β levels decreased in the presence of Δ2–18 WT and Δ2–18 P301L compared with full-length WT and P301L tau (WT tau, 0.0393 ± 0.0067; Δ2–18 WT, 0.1525 ± 0.0281; P301L, 0.0162 ± 0.0062; Δ2–18 P301L, 0.1231 ± 0.0125). I, WT tau, Δ2–18 WT, P301L, and Δ2–18 P301L in cells coexpressing PP1γ. J, Quantification indicated similar levels of tau expression with all constructs (WT tau, 8.82 ± 1.27; Δ2–18 WT, 7.26 ± 1.57; P301L, 8.32 ± 2.32; Δ2–18 P301L, 6.96 ± 1.73). K, Immunoblots of total PP1 (green) and inactive PP1γ (red, pT320 PP1 antibody corresponds to pT311 in PP1γ). L, The Δ2–18 WT resulted in decreased levels of active PP1γ compared with full-length counterparts (WT tau, 0.0578 ± 0.0099; Δ2–18 WT, 0.1428 ± 0.0154; P301L, 0.0101 ± 0.0022; Δ2–18 P301L, 0.0862 ± 0.0471). P301L tau expression led to higher levels of active PP1 compared with WT tau, and this effect was reduced about ninefold on PAD deletion in Δ2–18 P301L. Tau signal was normalized to GAPDH loading control (B, F, J) and pT320 PP1 signal was normalized to total PP1 signal (D, H, L). Data in legend are reported in ratios of the signal intensities in arbitrary units. All data in figure are mean ± SD and were compared using repeated measures one-way ANOVAs with a Geisser–Greenhouse correction and Tukey's post hoc multiple comparison test, *p ≤ 0.05).