pcas9 mcherry empty  (Addgene inc)

Journal: Science advances

Article Title: Actin-binding protein filamin-A drives tau aggregation and contributes to progressive supranuclear palsy pathology.

doi: 10.1126/sciadv.abm5029

Figure Lengend Snippet: Fig. 3. FLNA enhances the phosphorylation, protein stability and sarkosyl-insolubility of tau. (A) Immunoblotting (IB) of the lysates from HEK293 cells shows FLNAWT induces phosphorylation of GFP–4R-tau at the epitopes recognized by the two phosphorylated tau antibodies AT8 (Ser202/Thr205) and PHF-1 (Ser396/Ser404). (B) IB of the lysates from CHX chase assay in HEK293 cells shows that the GFP–4R-tau protein levels at 24 hours are significantly higher when FLNA are coexpressed compared to empty (n = 3). The levels of GFP–4R-tau are normalized to those of GAPDH at 0 hour. (C) IB of the lysates from HEK293 cells shows that overexpression of FLNAWT in HEK293 cells increases GFP–4R-tau concentration in homogenates (Ho), S1, and P3 (n = 3). (D) Immunoprecipitation (IP) assay. More FLNAWT proteins are coimmunoprecipitated with TAU-5 from lysates of HEK293 coexpressing GFP–4R-tau and FLNA. More HSP90, HSP70, HSP40, and ubiquitin are also coimmunoprecipitated. (E) Immunofluorescence shows AT8-immunopositive tau aggregates (blue) in the astrocytes coexpressing GFP–4R-tau (green) and mCherry-tagged FLNA (red) (FLNA+) but not in the astrocytes expressing sole GFP–4R-tau (FLNA−). (F) IB of homogenates from the astrocytes shows that FLNAWT significantly increases the protein levels of GFP–4R-tau with AT8-immunopositive hyperphosphorylation (n = 3). Arrow indicates nonspecific band. Scale bars, 10 m. Values are presented as means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05. Statistics obtained from Tukey-Kramer tests in (C) and Student’s t tests in (F).

Article Snippet: We generated expression vectors encoding wild-type FLNA (mCherryFLNAWT), p.Ala39Gly mutant FLNA (mCherry-FLNAAla39Gly), N-terminal trancated FLNA (mCherry-FLNAABD + Ig1–15), C-terminal trancated FLNA (mCherry-FLNAIg16–24), Ig1-8 domain of FLNA (mCherryFLNAIg1–8), Ig9-15 domain of FLNA (mCherry-FLNAIg9–15), Ig16-23 domain of FLNA (mCherry-FLNAIg16-23), truncated version of FLNA deleting Ig16-23 (mCherry-FLNAABD + Ig1-15+ Ig24), truncated version of FLNA deleting Ig1-8 and Ig16-23 (mCherry-FLNAABD + Ig9-15+ Ig24: FLNA), p.Ser2523Asn rare variant FLNA (mCherry-FLNASer2523Asn), p.Arg2334Cys rare variant FLNA (mCherry-FLNAArg2334Cys), p.Val2191Met rare variant FLNA (mCherry-FLNAVal2191Met), p.Ala2075Ser rare variant FLNA (mCherry-FLNAAla2075Ser), p.Arg2003His rare variant FLNA (mCherry-FLNAArg2003His), p.Leu1980Val rare variant FLNA (mCherryFLNALeu1980Val), p.Ser2523Ala non-phosphorylated variant FLNA (mCherry-FLNASer2523Ala), and mCherry (mCherry-empty) from mCherry-FilaminA-N-9 (Addgene, plasmid 55047) for in vitro assays.

Techniques: Phospho-proteomics, Western Blot, Over Expression, Concentration Assay, Immunoprecipitation, Ubiquitin Proteomics, Immunofluorescence, Expressing

Journal: Science advances

Article Title: Actin-binding protein filamin-A drives tau aggregation and contributes to progressive supranuclear palsy pathology.

doi: 10.1126/sciadv.abm5029

Figure Lengend Snippet: Fig. 4. Induction of human FLNA by electroporation enhances GFP–4R-tau protein levels through F-actin in murine brains. (A) Immunofluorescence for the E18 mouse brains that were electroporated with the indicated plasmids at E14 (n = 15). The electroporation of FLNAWT (red), but not mutant FLNA (FLNAAla39Gly) that abrogates the binding ability to F-actin, results in heterotopia (arrow) and a higher immunoreactivity for GFP–4R-tau (green) compared to empty. The immunoreactivity is normal- ized to the count of the mCherry-labeled electroporated cells. Scale bars, 100 m. (B) Immunofluorescence for primary cortical neurons from E15 brain that were electro- porated with the indicated plasmids at E14, followed by the treatment of 0.1% dimethyl sulfoxide (DMSO) or 20 nM cytochalasin D (CytoD) (n = 15). Overexpression of FLNAWT (red) with DMSO shows AT8-immunopositive tau aggregates (arrows) and significantly higher area ratio of AT8 (blue) to GFP (green) (% AT8/GFP) on 2 days in vitro (DIV) compared to empty; this phenomenon was attenuated with CytoD treatment. Phalloidin stain (magenta) was used to identify F-actin. Scale bars, 10 m. (C) Immunofluorescences for the P7 brains that were electroporated with the indicated plasmids at E14. The neurons, oligodendrocytes, and astrocytes in the P7 brains show accumulations of GFP–4R-tau (green) with FLNAWT (red). Anti-GFAP antibody (blue) was used to identify astrocytes. Scale bars, 10 m. (D) Immunoblotting (IB) of lysates from the P7 brains with the IUE shows that FLNAWT significantly increases the protein levels of GFP–4R-tau compared to empty in S1 and P3 (n = 3). Values are presented as means ± SEM. N.S., not significant. ***P < 0.001, **P < 0.01, and *P < 0.05. Statistics obtained from Tukey-Kramer tests in (A) and (B) and Student’s t tests in (D).

Article Snippet: We generated expression vectors encoding wild-type FLNA (mCherryFLNAWT), p.Ala39Gly mutant FLNA (mCherry-FLNAAla39Gly), N-terminal trancated FLNA (mCherry-FLNAABD + Ig1–15), C-terminal trancated FLNA (mCherry-FLNAIg16–24), Ig1-8 domain of FLNA (mCherryFLNAIg1–8), Ig9-15 domain of FLNA (mCherry-FLNAIg9–15), Ig16-23 domain of FLNA (mCherry-FLNAIg16-23), truncated version of FLNA deleting Ig16-23 (mCherry-FLNAABD + Ig1-15+ Ig24), truncated version of FLNA deleting Ig1-8 and Ig16-23 (mCherry-FLNAABD + Ig9-15+ Ig24: FLNA), p.Ser2523Asn rare variant FLNA (mCherry-FLNASer2523Asn), p.Arg2334Cys rare variant FLNA (mCherry-FLNAArg2334Cys), p.Val2191Met rare variant FLNA (mCherry-FLNAVal2191Met), p.Ala2075Ser rare variant FLNA (mCherry-FLNAAla2075Ser), p.Arg2003His rare variant FLNA (mCherry-FLNAArg2003His), p.Leu1980Val rare variant FLNA (mCherryFLNALeu1980Val), p.Ser2523Ala non-phosphorylated variant FLNA (mCherry-FLNASer2523Ala), and mCherry (mCherry-empty) from mCherry-FilaminA-N-9 (Addgene, plasmid 55047) for in vitro assays.

Techniques: Electroporation, Immunofluorescence, Mutagenesis, Binding Assay, Labeling, Over Expression, In Vitro, Staining, Western Blot