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. 2019 Oct 15;12:242. doi: 10.3389/fnmol.2019.00242

Figure 3.

Figure 3

(A) WT-Tau interacts with PARN, and the interaction is reduced by tau phosphorylation at pathological sites. Immobilized GST-WT-Tau or GST-PH-Tau, a tau variant with phosphorylation at pathological sites, on glutathione beads were incubated with His-PARN (top panel). Alternatively, immobilized His-PARN on nickel beads was incubated with GST-WT-Tau or GST-PH-Tau (bottom panel). Tau variants and His-PARN constructs were previously described (Alonso et al., 2010; Cevher et al., 2010). A representative pull-down reaction from three independent assays is shown. Equivalent amounts of the pulldowns (PD) and supernatants (SN) were analyzed and proteins were detected by immunoblotting with the indicated antibodies. Five percent of the proteins used in the PD assays are shown as input. On the left, relative binding (RB) of PH-Tau/His-PARN and WT-Tau/His-PARN was plotted. Binding of WT-Tau to His-PARN was arbitrarily set at 1.0. The data used are the mean RB ± SEM from three independent biological assays. Quantifications were done using ImageJ software (http://rsb.info.nih.gov/ij/). The P-values are indicated as **(≤0.005). (B) Immobilized His-PARN on nickel beads was incubated with NEs from UV-treated or untreated HCT116 cells. A representative pull-down reaction from three independent assays is shown. Equivalent amounts of the pulldowns (PD) and supernatants (SN) were analyzed and proteins were detected by immunoblotting with the indicated antibodies. Five percent of the NE used in the pull-down reactions is shown as input. (C) Coronal slices of hippocampus (CA3) stained with polyclonal PARN antibodies (red) showed changes in PARN distribution in mice expressing human PH-Tau (green). Non-transgenic mice (a,b) and WT-Tau expressing mice (e,f) showed PARN staining mainly associated to the nucleus, whereas the mice expressing human PH-Tau (c,d) showed accumulation of PARN in the perinuclear area co-localizing with PH-Tau. (a,c,e): only PARN staining; (b,d,f): merged images (PARN, PH-Tau and DAPI for the nuclei). Monoclonal human tau antibodies were used.