gst tagged hp1γ  (Addgene inc)

Journal: bioRxiv

Article Title: Tau interactions with inner nuclear envelope proteins modulates chromatin

doi: 10.64898/2025.12.05.692577

Figure Lengend Snippet: (A) Co-immunoprecipitation of SUN1 and LBR with GFP-Tau in SH-SY5Y cell lysates using high-salt buffer for cell lysis (left). (B) Principle of proximity ligation assays (PLA) for in situ detection of proteins in close proximity (<40 nm). (C) Positive and negative controls for endogenous Tau PLAs in untransfected SH-SY5Y cells. The negative control (Tau+IgG) shows few unspecific fluorescent PLA signal spots, whereas the Tau positive control using two Tau antibodies (Tau-13+Tau DAKO) results in high PLA signal throughout the cell body. The positive control for nuclear envelope localization, LaminA/C+SUN1, leads to PLA signal around the nucleus. Scale bars = 10 µm. (D) PLA of endogenous Tau with NE transmembrane proteins in SH-SY5Y cells. Quantification of PLA signal per cell nucleus validate SUN1 and Nesprin-2 as interactors of Tau. PLA signal was quantified in nuclear ROIs. n = 200-400. One-way ANOVA with Dunnett multiple comparison test. Scale bars = 10 µm. (E) PLA of endogenous Tau with inner NE proteins in SH-SY5Y cells. Quantification of PLA signal per nucleus validates Emerin and LBR as interactors of Tau. PLA signal was quantified in nuclear ROIs. n = ∼200. One-way ANOVA with Dunnett multiple comparison test. Scale bars = 10 µm. (F) PLA of endogenous Tau with intranuclear proteins in SH-SY5Y cells. Quantification of PLA signal per nucleus validates HP1γ as interactor of Tau. PLA signal was quantified in nuclear ROIs. n = ∼300. One-way ANOVA with Dunnett multiple comparison test. Scale bars = 10 µm. (G) PLA of endogenous Tau in mouse hippocampal neurons (DIV12). The negative Control (Tau+IgG) leads to few unspecific fluorescent spots, whereas Tau positive control (Tau-5+Tau DAKO) results in high PLA signal throughout the cell body. PLA signal was quantified in nuclear ROIs. Note that strong neuronal PLA signal is produced for Tau+LBR and Tau+HP1γ, but not for MAP2+LBR and MAP2+HP1γ. n =∼50. One-way ANOVA with Dunnett multiple comparison test. Scale bars = 10 µm. (H) Representative images of immunofluorescently labeled Tau interactors (SUN1, LBR, LEM2, HP1γ) in human AD brain sections (SUN1, LBR, HP1γ: hippocampal CA1; LEM2: cortex). Zoom-ins show selected neuronal nuclei/cell bodies (MAP2+) having different phospho-Tau (pS202/pT205/pT231) levels and aggregation states. Note that LBR seems to co-aggregate with phospho-Tau in neurons (arrow heads in zoom-ins). Scale bars = 10 µm. (I) Immunostaining for MAP2 (turquoise) and total Tau (pink) in combination with PLAs (yellow) for endogenous total Tau with SUN1 (left column 1), LBR (colum 2), LEM2 (column 3), and HP1γ (right column 4) in human AD brain sections (hippocampal CA1 and dentate gyrus). For each condition, white squares indicate location of zoom-ins shown below for combined IHC+PLA and PLA with DAPI. Bottom: PLA for Human brain negative controls (SUN1 only, SUN1+PSD-95) show no PLA signal. Scale bars = 10 µm.

Article Snippet: GST-tagged HP1γ (Addgene: #24076) was expressed in E. coli BL21 Star (DE3) (Invitrogen).

Techniques: Immunoprecipitation, Lysis, Ligation, In Situ, Negative Control, Positive Control, Comparison, Produced, Labeling, Immunostaining

Journal: bioRxiv

Article Title: Tau interactions with inner nuclear envelope proteins modulates chromatin

doi: 10.64898/2025.12.05.692577

Figure Lengend Snippet: (A) Schematics of the inner nuclear membrane protein LBR and its direct or HP1γ-mediated interaction with DNA via the N-terminal nuclear projection domain (npd). (B) Predictions of protein disorder along the peptide sequences of LBR, HP1γ and Tau using PONDR (Predictor of Natural Disordered Regions) reveal disordered regions (PONDR > 0.5) in LBR-npd, in the central region of HP1γ, and the disorder of Tau’s N-terminal projection domain. (C) PONDR vs. CatGranule score (algorithm to predict liquid-liquid phase separation propensity) of identified nuclear Tau interactors shows their disorder (PONDR > 0.5) and/or LLPS propensity (CatGranule > 0.75; http://s.tartaglialab.com/update_submission/902192/d53f367eaf ). Note that LEM2-npd has a high disorder/LLPS propensity. (D) LBR-npd (including 1% LBR-npd-Dylight488) condensation in the presence and absence of HP1γ (including 1% HP1γ-Dylight555), Tau (including 2% Tau-Dylight650), or both under the indicated conditions. Left: in the absence of DNA, LBR-npd readily forms grape-like assemblies of spherical condensates, into which both HP1γ and Tau co-partition. In the presence of λ-DNA (right), LBR-npd attaches to DNA and forms condensates that bundle DNA. HP1γ joins LBR-npd on thin DNA threads as well as in thicker condensates, whereas Tau is only found together with condensed LBR-npd on DNA. Scale bars = 10 µm. (E) Control conditions showing no condensation of HP1γ, Tau, or both with and without λ-DNA in the used assay buffer conditions. Scale bars = 10 µm. (F) FRAP of LBR-npd reveals liquid-like recovery in the absence of DNA, whereby HP1γ and Tau reduce the mobile fraction of LBR-npd both independently and co-cooperatively. When bound to λ-DNA, LBR-npd shows almost no recovery regardless of the absence or presence of HP1γ and/or Tau. LBR-npd bound to DNA shows no recovery in any condition. Data shown as mean±SD, n=10-15 condensates per condition from 3 experiments. One-way ANOVA with Tukey post-test was applied to compare the average of the last 10 mean values of each condition. Images show example FRAP images, Scale bars = 1 µm. (G) Electrophoretic mobility shift assays (EMSA; non-denaturing PAGE on 6% DNA retardation gels) of LBR-npd (400 ng) binding to DNA (500 bp, 20 ng) in the absence and presence (25, 50, or 75 ng Tau) of Tau. DNA was visualized by SYBR® Green EMSA Nucleic Acid Gel Stain. An upshift of signal in the gel lanes indicates that the DNA is bound by added proteins. Quantification of unbound DNA (lower lane parts, grey rectangle) versus LBR-npd/Tau bound DNA (upper lane parts, blue/cyan rectangle) was done based on densitometry. Black circles indicate conditions of comparable LBR-npd concentrations with and without 25 ng Tau.

Article Snippet: GST-tagged HP1γ (Addgene: #24076) was expressed in E. coli BL21 Star (DE3) (Invitrogen).

Techniques: Membrane, Control, Electrophoretic Mobility Shift Assay, Binding Assay, SYBR Green Assay, Staining