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Addgene inc plasmids cbh v5 aav9 abe n terminus

Intrathoracic ABE treatment corrects Tnni3 R193H mutation in cardiac tissue (A and B) Schematic illustration of the dual <t>AAV9</t> vector construction strategy and ABE treatment, administered via intrathoracic injections in 6-week-old Tnni3 R193H/R193H mice. (C and D) Efficient correction of Tnni3 mutation in the heart. Assessment of gene editing efficiency in the hearts of Tnni3 R193H/R193H mice 12 weeks after the AAV treatment. A>G editing efficiencies were measured in genomic DNA and mRNA using high-throughput sequencing. Each point represents an individual mouse. ∗∗∗∗ p < 0.0001 based on Student’s unpaired t test analysis. Data are presented as mean ± SEM. (E and F) Lack of liver editing following <t>the</t> <t>AAV9-ABE</t> treatment. As in (C), gene editing efficiency in the livers of Tnni3 R193H/R193H mice after 12 weeks of dual AAV treatment. A>G editing efficiency was also assessed in the genomic DNA and mRNA extracted from liver tissue, demonstrating diminished delivery and expression of the ABE system in the hepatic tissue. Each point represents an individual mouse. ns, not statistically significant (Student’s unpaired t test analysis). Data are presented as mean ± SEM.

Plasmids Cbh V5 Aav9 Abe N Terminus, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Therapeutic base editing alleviates restrictive cardiomyopathy"

Article Title: Therapeutic base editing alleviates restrictive cardiomyopathy

Journal: Cell Reports Medicine

doi: 10.1016/j.xcrm.2026.102639

Intrathoracic ABE treatment corrects Tnni3 R193H mutation in cardiac tissue (A and B) Schematic illustration of the dual AAV9 vector construction strategy and ABE treatment, administered via intrathoracic injections in 6-week-old Tnni3 R193H/R193H mice. (C and D) Efficient correction of Tnni3 mutation in the heart. Assessment of gene editing efficiency in the hearts of Tnni3 R193H/R193H mice 12 weeks after the AAV treatment. A>G editing efficiencies were measured in genomic DNA and mRNA using high-throughput sequencing. Each point represents an individual mouse. ∗∗∗∗ p < 0.0001 based on Student’s unpaired t test analysis. Data are presented as mean ± SEM. (E and F) Lack of liver editing following the AAV9-ABE treatment. As in (C), gene editing efficiency in the livers of Tnni3 R193H/R193H mice after 12 weeks of dual AAV treatment. A>G editing efficiency was also assessed in the genomic DNA and mRNA extracted from liver tissue, demonstrating diminished delivery and expression of the ABE system in the hepatic tissue. Each point represents an individual mouse. ns, not statistically significant (Student’s unpaired t test analysis). Data are presented as mean ± SEM.

Figure Legend Snippet: Intrathoracic ABE treatment corrects Tnni3 R193H mutation in cardiac tissue (A and B) Schematic illustration of the dual AAV9 vector construction strategy and ABE treatment, administered via intrathoracic injections in 6-week-old Tnni3 R193H/R193H mice. (C and D) Efficient correction of Tnni3 mutation in the heart. Assessment of gene editing efficiency in the hearts of Tnni3 R193H/R193H mice 12 weeks after the AAV treatment. A>G editing efficiencies were measured in genomic DNA and mRNA using high-throughput sequencing. Each point represents an individual mouse. ∗∗∗∗ p < 0.0001 based on Student’s unpaired t test analysis. Data are presented as mean ± SEM. (E and F) Lack of liver editing following the AAV9-ABE treatment. As in (C), gene editing efficiency in the livers of Tnni3 R193H/R193H mice after 12 weeks of dual AAV treatment. A>G editing efficiency was also assessed in the genomic DNA and mRNA extracted from liver tissue, demonstrating diminished delivery and expression of the ABE system in the hepatic tissue. Each point represents an individual mouse. ns, not statistically significant (Student’s unpaired t test analysis). Data are presented as mean ± SEM.

Techniques Used: Mutagenesis, Plasmid Preparation, Next-Generation Sequencing, Expressing

Sequencing:

Article Title: Therapeutic base editing alleviates restrictive cardiomyopathy.
Article Snippet: .. REAGENT or RESOURCE SOURCE IDENTIFIER Oligonucleotides See Table S1 for sgRNA sequence This paper N/A See Table S2 for oligos used in genotyping, RT-qPCR, and NGS This paper N/A Recombinant DNA Lenti-V2-Tnni3 (c.578G>A) This paper N/A AAV9-cTnT-SpRY HF1-ABE8e-N ter This paper N/A AAV9-cTnT-SpRY HF1-ABE8e-C ter This paper N/A pLX-sgRNAs This paper N/A Cbh_v5 AAV-ABE N-terminal Addgene Cat137177 Cbh_v5 AAV-ABE C-terminal Addgene Cat#137178 Software and algorithms GraphPad Prism 10 GraphPad http://www.graphpad.com/ HISAT2 v2.0.5 HISAT2 https://daehwankimlab.github.io/hisat2/ GSVA, v2.0.7 GSVA https://github.com/rcastelo/GSVA R (v4.4.2) R code http://www.r-project.org/ NRecon SkyScan Bruker microCT 2012-16, Version: 1.7.3.1 cTAn CT Analyzer Version: 1.17.7.2+ (64-bit) cTvol Realistic 3D-Visualization Version: 2.3.2.0 (64-bit) Cell Reports Medicine 7, 102639, March 17, 2026 e2 OPEN ACCESS Generation of Tnni3 R193H/R193H mice To evaluate the pathogenicity of the homozygous p.R192H variant in the TNNI3 gene, we generated a CRISPR/Cas9-mediated transgenic mouse model harboring the corresponding mutation p.R193H. .. The Tnni3 R193H knock-in model was created on a C57BL/6 J mouse strain (JAX #000664).

Article Title: Base editing rescues seizures and sudden death in a SCN8A mutation-associated developmental epileptic encephalopathy model
Article Snippet: .. AAV vector backbone plasmids were cloned to insert the sgRNA sequence and C-terminal base editor half of ABE8e-NRCH (V106W) into v5 Cbh-AAV-ABE-NpuC+U6-sgRNA (Addgene 137177), and the N-terminal base editor half v5 Cbh-AAV-ABE-NpuN (Addgene 137178) (Genscript Inc.) ( ). .. AAV vector backbone plasmids were cloned to insert the sgRNA sequence and C-terminal base editor half of ABE8e-NRCH (V106W) into v5 Cbh-AAV-ABE-NpuC+U6-sgRNA (Addgene 137177), and the N-terminal base editor half v5 Cbh-AAV-ABE-NpuN (Addgene 137178) (Genscript Inc.) ( ).

Next-Generation Sequencing:

Recombinant:

Bioprocessing:

Article Title: Therapeutic base editing alleviates restrictive cardiomyopathy.
Article Snippet: Mouse strains and husbandry conditions The Tnni3 R193H knock-in mouse model was generated on a C57BL/6 J genetic background (The Jackson Laboratory, #000664) through the services of Cyagen Biosciences Inc. .. The plasmids Cbh_v5 AAV9-ABE N-terminus (Addgene plasmid no. 137177) and Cbh_v5 AAV9-ABE C-terminus (Addgene plasmid no. 137178) were employed for AAV plasmid construction. .. DNA polymerase KOD One PCR Master Mix (Toyobo; KMM-101) and Uniclone One Step Seamless Cloning Kit (Genesand; SC612) were used.

Article Title: Precise gene editing of pathogenic Lamin A mutations corrects cardiac disease.
Article Snippet: For the in vitro editing experiments, the following plasmids were used: ABE8e (Addgene, 138489) (15), NG- ABE8e (Addgene, 138491) (15), pCMV_ABEmax_P2A_GFP (Addgene, 112101) (16), NG- ABEmax (Addgene, 124163) (17), pBT375 (Addgene, 125616) (18), all gifts from David Liu (Broad Institute of Harvard and MIT, Cambridge, MA); and pCAG- CBE4maxSpRY- P2A- EGFP (Addgene, 139999) (19), a gift from Benjamin Kleinstiver (Massachusetts General Hospital, Boston, MA). .. The N- terminal and C- terminal ABE and CBE constructs were adapted from Cbh_v5 AAV- ABE N- terminal (Addgene, 137177) (20) and Cbh_v5 AAV- ABE C- terminal (Addgene, 137178) (20), both gifts from David Liu (Broad Institute of Harvard and MIT, Cambridge, MA), and from the aforementioned plasmids as previously described in our laboratory (21). .. Cloning was performed using NEBuilder HiFi DNA Assembly Cloning Kit (NEB, E5520S).

Article Title: Therapeutic base editing alleviates restrictive cardiomyopathy
Article Snippet: .. Cbh_v5 AAV-ABE N-terminal , Addgene , Cat137177. .. Cbh_v5 AAV-ABE C-terminal , Addgene , Cat#137178.

Article Title: Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice
Article Snippet: Expression plasmids for human U6 promoter-driven gRNAs were generated by annealing and ligating duplexed oligonucleotides corresponding to spacer sequences into BsmBI-digested pUC19-U6-BsmBI_cassette-SpCas9_gRNA (BPK1520; Addgene plasmid 65777). .. Npu intein-split ABE constructs were cloned into N- and C-terminal AAV plasmids (Addgene plasmids 137177 and 137178, respectively). ..

Software:

Variant Assay:

Generated:

CRISPR:

Transgenic Assay:

Mutagenesis:

Plasmid Preparation:

Construct:

other:

Article Title: Therapeutic base editing alleviates restrictive cardiomyopathy
Article Snippet: To generate a stable cell line harboring the Tnni3 (c.578G>A) mutation, HEK 293 T cells (ATCC, the cell lines have not been authenticated) were transduced with a lentiviral lentiCRISPRv2 vector (pLenti-V2) carrying the mutant Tnni3 sequence.

Clone Assay:

Image Search Results

Aggregated phospho-S670-GRK2 on dysfunctional mitochondria of aged AD mice (A) Immunoblot of GRK2 in hippocampal lysates of 18-month-old Tg2576 (AD) and B6 mice. GNAO served as loading control. The lower panel shows quantitative data. (B and C) Immunofluorescence of GRK2 (green) and mitochondrial TOMM40 (red) in the CA1 hippocampal area of 18-month-old Tg2576 (left) and B6 (right) mice. Upper panels show the area marked with a white square frame in a 5-fold higher magnification (scale bars: 20 μm). Nuclei are stained with DAPI (blue). Data are from n = 4 mice per group. Replicates are shown in A and S1B. Quantitative data are shown in (C) and E–S1H. (D–G) Immunoblot of hippocampal cytosolic (D and F) and mitochondrial (E and G) GRK2 and phospho-S670-GRK2 (p-S670) in 18-month-old Tg2576 (AD-18mo), 18-month-old B6 (B6-18mo), and 12-month-old Tg2576 (AD-12mo) mice. Cytosolic PPP2CA was determined (D and F). ADSL and TOMM40 are loading controls. (H) Immunoblot of hippocampal mitochondrial Aβ (IB: Aβ) in 18-month-old Tg2576 (AD-18mo), B6 (B6-18mo), and 12-month-old Tg2576 (AD-12mo) mice. Arrowheads mark monomeric Aβ peptides and APP. TOMM40 is the loading control. (I and J) Hippocampal mitochondrial ACO2 and reactivated ACO2 activity (I) and mitochondrial ATP (J) of 12- and 18-month-old Tg2576 and B6 mice. Data are mean ± SD; n = 4 mice per group (A, C, D, F, I, and J) or n = 4 biological replicates (E, G, and H; hippocampi from three mice were pooled for one isolation, which is defined as a biological replicate); unpaired, two-tailed t test [df = 6; t = 9.179 (A); 3.613, 1.357, 0.3557, 5.674 (C); 3.833, 22.22, 10.66 (D); 6.815, 2.083, 9.466, 2.817 (E); 2.806, 11.00, 4.495 (F); 11.47, 18.28, 11.65, 23.38 (G)]; one-way ANOVA and Tukey’s test [F(2,9) = 55.77 (H); F(3,12) = 113.9, 12.37 (I); 21.54 (J)]. See also A, S1B, S1E–S1H, and .

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: Aggregated phospho-S670-GRK2 on dysfunctional mitochondria of aged AD mice (A) Immunoblot of GRK2 in hippocampal lysates of 18-month-old Tg2576 (AD) and B6 mice. GNAO served as loading control. The lower panel shows quantitative data. (B and C) Immunofluorescence of GRK2 (green) and mitochondrial TOMM40 (red) in the CA1 hippocampal area of 18-month-old Tg2576 (left) and B6 (right) mice. Upper panels show the area marked with a white square frame in a 5-fold higher magnification (scale bars: 20 μm). Nuclei are stained with DAPI (blue). Data are from n = 4 mice per group. Replicates are shown in A and S1B. Quantitative data are shown in (C) and E–S1H. (D–G) Immunoblot of hippocampal cytosolic (D and F) and mitochondrial (E and G) GRK2 and phospho-S670-GRK2 (p-S670) in 18-month-old Tg2576 (AD-18mo), 18-month-old B6 (B6-18mo), and 12-month-old Tg2576 (AD-12mo) mice. Cytosolic PPP2CA was determined (D and F). ADSL and TOMM40 are loading controls. (H) Immunoblot of hippocampal mitochondrial Aβ (IB: Aβ) in 18-month-old Tg2576 (AD-18mo), B6 (B6-18mo), and 12-month-old Tg2576 (AD-12mo) mice. Arrowheads mark monomeric Aβ peptides and APP. TOMM40 is the loading control. (I and J) Hippocampal mitochondrial ACO2 and reactivated ACO2 activity (I) and mitochondrial ATP (J) of 12- and 18-month-old Tg2576 and B6 mice. Data are mean ± SD; n = 4 mice per group (A, C, D, F, I, and J) or n = 4 biological replicates (E, G, and H; hippocampi from three mice were pooled for one isolation, which is defined as a biological replicate); unpaired, two-tailed t test [df = 6; t = 9.179 (A); 3.613, 1.357, 0.3557, 5.674 (C); 3.833, 22.22, 10.66 (D); 6.815, 2.083, 9.466, 2.817 (E); 2.806, 11.00, 4.495 (F); 11.47, 18.28, 11.65, 23.38 (G)]; one-way ANOVA and Tukey’s test [F(2,9) = 55.77 (H); F(3,12) = 113.9, 12.37 (I); 21.54 (J)]. See also A, S1B, S1E–S1H, and .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Western Blot, Control, Immunofluorescence, Staining, Activity Assay, Isolation, Two Tailed Test

TOMM6 is an interaction partner of phospho-S670-GRK2 (A) Immunoblot of GRK2 in brain lysates of 18- and 12-month-old Tg2576 AD mice. Data are mean ± SD; n = 4 mice; (unpaired, two-tailed t test; df = 6, t = 20.02). (B and C) Immunoaffinity enrichment of phospho-S670-GRK2 (AP: p-S670, lanes 1–4) from 18-month-old (B; lanes 1 and 2) and 12-month-old (C; lanes 3 and 4) Tg2576 mouse brain mitochondria followed by immunoblot detection (IB) of enriched phospho-S670-GRK2 (left) and co-enriched TOMM6 (right). Lane 5 (C) shows a control immuno-affinity matrix (con). Two biological replicates are from 18-month-old mice, and three biological replicates are from 12-month-old Tg2576 mice. (D) Immunoaffinity enrichment of phospho-S670-GRK2 (AP: p-S670-GRK2) from brain mitochondria of 12-month-old Tg2576 mice and nano-LC-ESI-MS/MS identification of co-enriched TOMM6. (E) Immunoblot (IB) of TOMM6 (upper) and TOMM40 (lower) on brain mitochondria from 18-month-old Tg2576 and B6 mice. The right panel shows quantitative data. (F) Immunoaffinity enrichment of TOMM6 (AP: TOMM6) from brain mitochondria of 18-month-old Tg2576 (AD) and B6 (B6) mice, followed by immunoblot detection (IB) of enriched TOMM6 (left) and co-enriched TOMM40 (middle). The right panel shows quantitative data. (E and F) Data are mean ± SD; n = 4 biological replicates; unpaired, two-tailed t test [df = 6; t = 0.06099 (E), 12.59 (F)]. (G and H) In vitro phosphorylation of TOMM6 by GRK2 or p-S670-GRK2 (pS670). TOMM6-ST-AA was not phosphorylated by GRK2. (H) shows quantitative data; mean ± SD; n = 4 biological replicates; one-way ANOVA and Tukey’s test; F(2,9) = 83.12, 206.5. (I) MDS data of TOMM6 showing the frequency distribution of the unfolded molecular weight (MW) of TOMM6 ( n = 40 measurements; left) and the linear regression analysis of the relationship between unfolded MW and TOMM6 (right). (J) Binding energies (E_RDOCK) of monomer-monomer docking, yielding dimers, and tetramer-tetramer docking yielding octamers, of wild-type TOMM6 (WT) and phosphomimetic TOMM6-ST-DD. Data represent means with range; one-way ANOVA and Tukey’s test [F(3,20) = 7.127]. (K) Immunoblot (IB) of TOMM6 in primary neurons expressing wild-type TOMM6 (WT) or TOMM6-ST-DD. Neurons were treated without and with aggregated Aβ and the GRK2 inhibitor, CMPD101 [+Aβ (immunoblot), +Aβ+101 (bar graph)]. Data represent mean ± SD; n = 4 biological replicates; one-way ANOVA and Tukey’s test [F(3,12) = 2141]. See also .

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: TOMM6 is an interaction partner of phospho-S670-GRK2 (A) Immunoblot of GRK2 in brain lysates of 18- and 12-month-old Tg2576 AD mice. Data are mean ± SD; n = 4 mice; (unpaired, two-tailed t test; df = 6, t = 20.02). (B and C) Immunoaffinity enrichment of phospho-S670-GRK2 (AP: p-S670, lanes 1–4) from 18-month-old (B; lanes 1 and 2) and 12-month-old (C; lanes 3 and 4) Tg2576 mouse brain mitochondria followed by immunoblot detection (IB) of enriched phospho-S670-GRK2 (left) and co-enriched TOMM6 (right). Lane 5 (C) shows a control immuno-affinity matrix (con). Two biological replicates are from 18-month-old mice, and three biological replicates are from 12-month-old Tg2576 mice. (D) Immunoaffinity enrichment of phospho-S670-GRK2 (AP: p-S670-GRK2) from brain mitochondria of 12-month-old Tg2576 mice and nano-LC-ESI-MS/MS identification of co-enriched TOMM6. (E) Immunoblot (IB) of TOMM6 (upper) and TOMM40 (lower) on brain mitochondria from 18-month-old Tg2576 and B6 mice. The right panel shows quantitative data. (F) Immunoaffinity enrichment of TOMM6 (AP: TOMM6) from brain mitochondria of 18-month-old Tg2576 (AD) and B6 (B6) mice, followed by immunoblot detection (IB) of enriched TOMM6 (left) and co-enriched TOMM40 (middle). The right panel shows quantitative data. (E and F) Data are mean ± SD; n = 4 biological replicates; unpaired, two-tailed t test [df = 6; t = 0.06099 (E), 12.59 (F)]. (G and H) In vitro phosphorylation of TOMM6 by GRK2 or p-S670-GRK2 (pS670). TOMM6-ST-AA was not phosphorylated by GRK2. (H) shows quantitative data; mean ± SD; n = 4 biological replicates; one-way ANOVA and Tukey’s test; F(2,9) = 83.12, 206.5. (I) MDS data of TOMM6 showing the frequency distribution of the unfolded molecular weight (MW) of TOMM6 ( n = 40 measurements; left) and the linear regression analysis of the relationship between unfolded MW and TOMM6 (right). (J) Binding energies (E_RDOCK) of monomer-monomer docking, yielding dimers, and tetramer-tetramer docking yielding octamers, of wild-type TOMM6 (WT) and phosphomimetic TOMM6-ST-DD. Data represent means with range; one-way ANOVA and Tukey’s test [F(3,20) = 7.127]. (K) Immunoblot (IB) of TOMM6 in primary neurons expressing wild-type TOMM6 (WT) or TOMM6-ST-DD. Neurons were treated without and with aggregated Aβ and the GRK2 inhibitor, CMPD101 [+Aβ (immunoblot), +Aβ+101 (bar graph)]. Data represent mean ± SD; n = 4 biological replicates; one-way ANOVA and Tukey’s test [F(3,12) = 2141]. See also .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Western Blot, Two Tailed Test, Control, Tandem Mass Spectroscopy, In Vitro, Phospho-proteomics, Molecular Weight, Binding Assay, Expressing

Inactive GRK2 enhances TOMM6 aggregation, Aβ plaques, and mortality, whereas wild-type GRK2 is beneficial (A) Immunoblot of hippocampal cytosolic GRK2 in 16-month-old Tg2576- GRK2S670D (S670D) and Tg2576 (AD16mo) mice (upper blot). The control blot detects ADSL (second blot). The third blot shows GRK2 in hippocampal lysates from 16-month-old Tg2576- GRK2S670D (S670D) and 16-month-old Tg2576- GRK2 (GRK2) mice. The lower control blot detects GNAO. (B) Immunoblot of hippocampal cytosolic GRK2 (upper) and p-S670-GRK2 (middle) in 16-month-old Tg2576- GRK2K220R (K220R), Tg2576- GRK2 (GRK2), and Tg2576- GRKInh (Inh) mice. ADSL is the cytosolic loading control. (A and B) Data represent mean ± SD; n = 4 mice per group; unpaired, two-tailed t test (A; df = 6, t = 5.640, 9.369, 4.242); one-way ANOVA and Tukey’s test [B; F(2,9) = 7.991, 31.84]. (C) Immunoblot of hippocampal mitochondrial GRK2 (upper) and mitochondrial TOMM6 (lower) in 16-month-old Tg2576- GRK2S670D (S670D) and Tg2576 (16 mo) mice. TOMM40 is a loading control (middle panel). (D–F) Immunoblot of hippocampal mitochondrial GRK2 (D and E) and TOMM6 (F) in 16-month-old Tg2576- GRK2K220R (K220R), Tg2576- GRK2 (GRK2), and Tg2576- GRKInh (Inh) mice. TOMM40 is the loading control (E, lower panel). (D and F) present quantitative data. (C, D, and F) Data are mean ± SD; n = 4 biological replicates; unpaired, two-tailed t test (C; df = 6, t = 7.403, t = 10.67, t = 4.247); one-way ANOVA and Tukey’s test [D,F; F(2,9) = 38.19, 137.0]. (G) Hippocampal mitochondrial ACO2 activity without (left) and with reactivation (right) in 16-month-old Tg2576- GRK2S670D (S670D), Tg2576- GRK2K220R (K220R), Tg2576- GRKInh (GRKInh), and Tg2576- GRK2 (GRK2) mice in comparison to 16-month-old Tg2576 (AD16mo) mice. Data are mean ± SD; n = 4 mice per group; one-way ANOVA and Tukey’s test; F(4,15) = 63.00 (left), 4.111 (right). (H) Probability of survival of the indicated transgenic mouse lines with Kaplan-Meier survival analysis and log rank (Mantel-Cox) test; n = 103 male and female mice ( n = 136 for Tg- GRK2K220R ); df = 1; chi-square 4.940 and p = 0.0264 for Tg2576- GRK2 vs. Tg2576; chi-square 16.83 and p < 0.0001 for Tg2576- GRK2S670D vs. Tg2576; df = 1; chi-square 20.70 and p < 0.0001 for Tg- GRK2K220R vs. Tg2576; chi-square 7.459 and p = 0.0063 for Tg2576- GRKInh vs. Tg2576; chi-square 28.51 and p < 0.0001 for Tg2576- GRK2K220R vs. Tg2576. (I–K) Hippocampal γ-secretase (I), β-secretase (J), and α-secretase (K) of 16-month-old Tg2576- GRK2S670D (S670D), Tg2576- GRK2K220R (K220R), Tg2576- GRKInh (GRKInh), and Tg2576- GRK2 (GRK2) mice compared to Tg2576 (AD16mo) mice. Data represent mean ± SD; n = 6 mice per group; one-way ANOVA and Tukey’s test; F(4,25) = 55.73 (I), 44.06 (J), 41.47 (K). (L) Aβ plaque load of histological sections shown in (M); E and C. Data are mean ± SD; n = 6 mice per group; one-way ANOVA and Tukey’s test [left panel; F(3,20) = 12.13]; unpaired, two-tailed t test (right panel; df = 10, t = 4.537). (M) Hippocampal Aβ plaques (brown) of 16-month-old Tg2576- GRK2S670D , Tg2576- GRK2K220R , Tg2576- GRKIn h, and Tg2576 (Tg2576 16mo) mice and 18-month-old Tg2576- GRK2 and Tg2576 (Tg2576 18mo) female (f) and male (m) mice. Counterstaining was performed with hematoxylin (HE). Immunohistology is representative of six mice per group; scale bars: 200 μm. Replicates are shown in E and C. See also and .

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: Inactive GRK2 enhances TOMM6 aggregation, Aβ plaques, and mortality, whereas wild-type GRK2 is beneficial (A) Immunoblot of hippocampal cytosolic GRK2 in 16-month-old Tg2576- GRK2S670D (S670D) and Tg2576 (AD16mo) mice (upper blot). The control blot detects ADSL (second blot). The third blot shows GRK2 in hippocampal lysates from 16-month-old Tg2576- GRK2S670D (S670D) and 16-month-old Tg2576- GRK2 (GRK2) mice. The lower control blot detects GNAO. (B) Immunoblot of hippocampal cytosolic GRK2 (upper) and p-S670-GRK2 (middle) in 16-month-old Tg2576- GRK2K220R (K220R), Tg2576- GRK2 (GRK2), and Tg2576- GRKInh (Inh) mice. ADSL is the cytosolic loading control. (A and B) Data represent mean ± SD; n = 4 mice per group; unpaired, two-tailed t test (A; df = 6, t = 5.640, 9.369, 4.242); one-way ANOVA and Tukey’s test [B; F(2,9) = 7.991, 31.84]. (C) Immunoblot of hippocampal mitochondrial GRK2 (upper) and mitochondrial TOMM6 (lower) in 16-month-old Tg2576- GRK2S670D (S670D) and Tg2576 (16 mo) mice. TOMM40 is a loading control (middle panel). (D–F) Immunoblot of hippocampal mitochondrial GRK2 (D and E) and TOMM6 (F) in 16-month-old Tg2576- GRK2K220R (K220R), Tg2576- GRK2 (GRK2), and Tg2576- GRKInh (Inh) mice. TOMM40 is the loading control (E, lower panel). (D and F) present quantitative data. (C, D, and F) Data are mean ± SD; n = 4 biological replicates; unpaired, two-tailed t test (C; df = 6, t = 7.403, t = 10.67, t = 4.247); one-way ANOVA and Tukey’s test [D,F; F(2,9) = 38.19, 137.0]. (G) Hippocampal mitochondrial ACO2 activity without (left) and with reactivation (right) in 16-month-old Tg2576- GRK2S670D (S670D), Tg2576- GRK2K220R (K220R), Tg2576- GRKInh (GRKInh), and Tg2576- GRK2 (GRK2) mice in comparison to 16-month-old Tg2576 (AD16mo) mice. Data are mean ± SD; n = 4 mice per group; one-way ANOVA and Tukey’s test; F(4,15) = 63.00 (left), 4.111 (right). (H) Probability of survival of the indicated transgenic mouse lines with Kaplan-Meier survival analysis and log rank (Mantel-Cox) test; n = 103 male and female mice ( n = 136 for Tg- GRK2K220R ); df = 1; chi-square 4.940 and p = 0.0264 for Tg2576- GRK2 vs. Tg2576; chi-square 16.83 and p < 0.0001 for Tg2576- GRK2S670D vs. Tg2576; df = 1; chi-square 20.70 and p < 0.0001 for Tg- GRK2K220R vs. Tg2576; chi-square 7.459 and p = 0.0063 for Tg2576- GRKInh vs. Tg2576; chi-square 28.51 and p < 0.0001 for Tg2576- GRK2K220R vs. Tg2576. (I–K) Hippocampal γ-secretase (I), β-secretase (J), and α-secretase (K) of 16-month-old Tg2576- GRK2S670D (S670D), Tg2576- GRK2K220R (K220R), Tg2576- GRKInh (GRKInh), and Tg2576- GRK2 (GRK2) mice compared to Tg2576 (AD16mo) mice. Data represent mean ± SD; n = 6 mice per group; one-way ANOVA and Tukey’s test; F(4,25) = 55.73 (I), 44.06 (J), 41.47 (K). (L) Aβ plaque load of histological sections shown in (M); E and C. Data are mean ± SD; n = 6 mice per group; one-way ANOVA and Tukey’s test [left panel; F(3,20) = 12.13]; unpaired, two-tailed t test (right panel; df = 10, t = 4.537). (M) Hippocampal Aβ plaques (brown) of 16-month-old Tg2576- GRK2S670D , Tg2576- GRK2K220R , Tg2576- GRKIn h, and Tg2576 (Tg2576 16mo) mice and 18-month-old Tg2576- GRK2 and Tg2576 (Tg2576 18mo) female (f) and male (m) mice. Counterstaining was performed with hematoxylin (HE). Immunohistology is representative of six mice per group; scale bars: 200 μm. Replicates are shown in E and C. See also and .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Western Blot, Control, Two Tailed Test, Activity Assay, Comparison, Transgenic Assay

Neuron-specific TOMM6 counteracts insoluble Aβ aggregation but increases soluble Aβ and mortality (A) Plasmid for generation of Tg2576- TOMM6 mice (upper) and PCR genotyping (middle and lower panels); c, control without genomic DNA; P, positive plasmid control; M, DNA marker. Double-transgenic mice are marked with an asterisk. (B–D) Immunoblot of hippocampal mitochondrial TOMM6 (B), GRK2 (C), and phospho-S670-GRK2 (D) of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. The control immunoblot (C, lower panel) detects TOMM40. (E and F) Immunoblot of hippocampal, cytosolic GRK2 (E, left) and phospho-S670-GRK2 (E, right) of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. The control blot detects ADSL. Quantitative data are shown in (F). (B, C, D, and F) Data are mean ± SD; n = 4 biological replicates per group (B, C, and D) and n = 4 mice per group (F); unpaired, two-tailed t test; df = 6; t = 4.741, 74.19 (B); 10.33, 13.47 (C); 7.329, 8.060 (D); 1.776, 0.3092 (F). (G) Immunofluorescence of GRK2 (green) and TOMM40 (red) in the hippocampal CA1 area of 18-month-old Tg2576 (left) and Tg2576- TOMM6 (right) mice. Nuclei are stained with DAPI (blue). The upper panels show the area marked by a white frame in a 5-fold higher magnification (scale bars: 20 μm). Immunofluorescence is representative of n = 4 mice per group. Replicates and quantitative data are shown in A, S1C, and S1E–S1H. (H and I) Hippocampal mitochondrial ACO2 activity (H) and mitochondrial ATP (I) of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. Data are mean ± SD ( n = 4 mice per group); unpaired, two-tailed t test; df = 6; t = 10.77, 3.346 (H); t = 4.420 (I). (J) Immunohistology of hippocampal Aβ plaques (brown) of 18-month-old Tg2576 and Tg2576- TOMM6 male (m) mice. Nuclei were stained with hematoxylin (HE). Immunohistology is representative of six mice per group (scale bar: 200 μm). Replicates and quantitative data are shown in A and S5B. (K and L) Insoluble (K) and soluble (L) Aβ1-40 and Aβ1-42 in brains of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. Data are mean ± SD; n = 6 mice per group; unpaired, two-tailed t test; df = 10; t = 12.59, 13.23 (K); 12.06, 9.365 (L). (M) Probability of survival of male and female Tg2576- TOMM6 ( n = 62), Tg2576 ( n = 103), and Tg- TOMM6 mice ( n = 103) was performed by Kaplan-Meier survival analysis with log rank (Mantel-Cox) test; df = 1, chi-square 12.52 and p = 0.0004 for Tg- TOMM6 vs. Tg2576; chi-square 11.7 and p = 0.0006 for Tg2576- TOMM vs. Tg2576. (N) Hippocampal α-, β-, and γ-secretase in 18-month-old Tg2576 and Tg2576- TOMM6 mice. Data represent mean ± SD; n = 6 mice per group (unpaired, two-tailed t test; df = 10, t = 1.519, 2.078, 1.791). See also A, S1C, S1E–S1H, and .

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: Neuron-specific TOMM6 counteracts insoluble Aβ aggregation but increases soluble Aβ and mortality (A) Plasmid for generation of Tg2576- TOMM6 mice (upper) and PCR genotyping (middle and lower panels); c, control without genomic DNA; P, positive plasmid control; M, DNA marker. Double-transgenic mice are marked with an asterisk. (B–D) Immunoblot of hippocampal mitochondrial TOMM6 (B), GRK2 (C), and phospho-S670-GRK2 (D) of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. The control immunoblot (C, lower panel) detects TOMM40. (E and F) Immunoblot of hippocampal, cytosolic GRK2 (E, left) and phospho-S670-GRK2 (E, right) of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. The control blot detects ADSL. Quantitative data are shown in (F). (B, C, D, and F) Data are mean ± SD; n = 4 biological replicates per group (B, C, and D) and n = 4 mice per group (F); unpaired, two-tailed t test; df = 6; t = 4.741, 74.19 (B); 10.33, 13.47 (C); 7.329, 8.060 (D); 1.776, 0.3092 (F). (G) Immunofluorescence of GRK2 (green) and TOMM40 (red) in the hippocampal CA1 area of 18-month-old Tg2576 (left) and Tg2576- TOMM6 (right) mice. Nuclei are stained with DAPI (blue). The upper panels show the area marked by a white frame in a 5-fold higher magnification (scale bars: 20 μm). Immunofluorescence is representative of n = 4 mice per group. Replicates and quantitative data are shown in A, S1C, and S1E–S1H. (H and I) Hippocampal mitochondrial ACO2 activity (H) and mitochondrial ATP (I) of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. Data are mean ± SD ( n = 4 mice per group); unpaired, two-tailed t test; df = 6; t = 10.77, 3.346 (H); t = 4.420 (I). (J) Immunohistology of hippocampal Aβ plaques (brown) of 18-month-old Tg2576 and Tg2576- TOMM6 male (m) mice. Nuclei were stained with hematoxylin (HE). Immunohistology is representative of six mice per group (scale bar: 200 μm). Replicates and quantitative data are shown in A and S5B. (K and L) Insoluble (K) and soluble (L) Aβ1-40 and Aβ1-42 in brains of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. Data are mean ± SD; n = 6 mice per group; unpaired, two-tailed t test; df = 10; t = 12.59, 13.23 (K); 12.06, 9.365 (L). (M) Probability of survival of male and female Tg2576- TOMM6 ( n = 62), Tg2576 ( n = 103), and Tg- TOMM6 mice ( n = 103) was performed by Kaplan-Meier survival analysis with log rank (Mantel-Cox) test; df = 1, chi-square 12.52 and p = 0.0004 for Tg- TOMM6 vs. Tg2576; chi-square 11.7 and p = 0.0006 for Tg2576- TOMM vs. Tg2576. (N) Hippocampal α-, β-, and γ-secretase in 18-month-old Tg2576 and Tg2576- TOMM6 mice. Data represent mean ± SD; n = 6 mice per group (unpaired, two-tailed t test; df = 10, t = 1.519, 2.078, 1.791). See also A, S1C, S1E–S1H, and .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Plasmid Preparation, Control, Marker, Transgenic Assay, Western Blot, Two Tailed Test, Immunofluorescence, Staining, Activity Assay

Development of a GRK2 function modulator that prevents phospho-S670-GRK2 and TOMM6 aggregation (A) CPD10 ligand affinity chromatography (AP:C10) with brain lysates of 12-month-old Tg2576- GRK2 mice. CPD10-interacting GRK2 (lanes 3 and 4; left) was eluted from the CPD10-containing affinity matrix and detected by immunoblot (IB). TOMM6 did not interact and was below detection limit (lanes 3 and 4; right). Lanes 1 and 2 show a control affinity matrix (AP: Con). (B) Enrichment of p-S670-GRK2(K220R) (lanes 7 and 8; left) and co-enrichment of TOMM6 (lanes 7 and 8; right) by CPD10 ligand affinity chromatography (AP:C10) from brains from 12-month-old Tg2576- GRK2K220R mice. Lanes 5 and 6 show a control affinity matrix. (A and B) Two biological replicates are shown for each transgenic mouse line. (C and D) Immunoaffinity enrichment (AP) of TOMM6 from brain mitochondria of 18-month-old Tg2576 mice without or with CPD10 treatment (±C10) for 6 months followed by immunoblot detection of enriched TOMM6 (IB: TOMM6) and phosphorylated TOMM6 (IB: p-Ser). Representative immunoblots (C) and quantitative data (D) are shown. Data represent mean ± SD; n = 4 biological replicates per group; unpaired, two-tailed t test; df = 6; t = 3.611 (TOMM6 aggr.), 11.74 (TOMM6 monomer), 2.811 (phospho-TOMM6 aggr.), 9.016 (phospho-TOMM6 monomer). (E) Molecular docking of CPD10 (CPK model, red) to GRK2 (upper) and to p-S670-GRK2 (middle) by CDOCKER. Left images show GRK2 and p-S670-GRK2 with modeled C-terminus (red), the large lobe (yellow), the small lobe (blue), and N-terminal and C-terminal domains of GRK2 (gray). The S670 phosphorylation is shown in green (CPK model). Right panels show the docking site of CPD10 in the large lobe of GRK2 (upper) and in the ATP-binding site of p-S670-GRK2 (middle). (F) Binding energies (kcal/mol) of CPD10 (S-isomer) docking to GRK2 and phospho-S670-GRK2. Data are mean of top five poses ± SD: −389.1 ± 12.1; +40.3 ± 32.7; −81.2 ± 30.3. (G) IC 50 values of CPD10 for inhibition of TOMM6 phosphorylation by p-S670-GRK2 and GRK2-S670A (S670A). Data are mean ± SD; n = 4 independent experiments with four technical replicates each. (H) CPD10 (10 μM) did not substantially inhibit GRK2-related kinases. (I) Conceptual model and molecular dynamics simulation of GRK2 function modulation by CPD10. CPD10 could stabilize the GRK2 monomer (cyan, right) and prevent phospho-S670-GRK2 and TOMM6 aggregation (left); OMM: outer mitochondrial membrane; IMS: intermembrane space. Potential energies (E) of GRK2 (lower right) and phospho-S670-GRK2 (p-S670; lower left) without (w/o) and with CPD10 (+C10) interaction are given after energy minimization without (w/o MD sim) and with MD simulation (MD sim). See also and .

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: Development of a GRK2 function modulator that prevents phospho-S670-GRK2 and TOMM6 aggregation (A) CPD10 ligand affinity chromatography (AP:C10) with brain lysates of 12-month-old Tg2576- GRK2 mice. CPD10-interacting GRK2 (lanes 3 and 4; left) was eluted from the CPD10-containing affinity matrix and detected by immunoblot (IB). TOMM6 did not interact and was below detection limit (lanes 3 and 4; right). Lanes 1 and 2 show a control affinity matrix (AP: Con). (B) Enrichment of p-S670-GRK2(K220R) (lanes 7 and 8; left) and co-enrichment of TOMM6 (lanes 7 and 8; right) by CPD10 ligand affinity chromatography (AP:C10) from brains from 12-month-old Tg2576- GRK2K220R mice. Lanes 5 and 6 show a control affinity matrix. (A and B) Two biological replicates are shown for each transgenic mouse line. (C and D) Immunoaffinity enrichment (AP) of TOMM6 from brain mitochondria of 18-month-old Tg2576 mice without or with CPD10 treatment (±C10) for 6 months followed by immunoblot detection of enriched TOMM6 (IB: TOMM6) and phosphorylated TOMM6 (IB: p-Ser). Representative immunoblots (C) and quantitative data (D) are shown. Data represent mean ± SD; n = 4 biological replicates per group; unpaired, two-tailed t test; df = 6; t = 3.611 (TOMM6 aggr.), 11.74 (TOMM6 monomer), 2.811 (phospho-TOMM6 aggr.), 9.016 (phospho-TOMM6 monomer). (E) Molecular docking of CPD10 (CPK model, red) to GRK2 (upper) and to p-S670-GRK2 (middle) by CDOCKER. Left images show GRK2 and p-S670-GRK2 with modeled C-terminus (red), the large lobe (yellow), the small lobe (blue), and N-terminal and C-terminal domains of GRK2 (gray). The S670 phosphorylation is shown in green (CPK model). Right panels show the docking site of CPD10 in the large lobe of GRK2 (upper) and in the ATP-binding site of p-S670-GRK2 (middle). (F) Binding energies (kcal/mol) of CPD10 (S-isomer) docking to GRK2 and phospho-S670-GRK2. Data are mean of top five poses ± SD: −389.1 ± 12.1; +40.3 ± 32.7; −81.2 ± 30.3. (G) IC 50 values of CPD10 for inhibition of TOMM6 phosphorylation by p-S670-GRK2 and GRK2-S670A (S670A). Data are mean ± SD; n = 4 independent experiments with four technical replicates each. (H) CPD10 (10 μM) did not substantially inhibit GRK2-related kinases. (I) Conceptual model and molecular dynamics simulation of GRK2 function modulation by CPD10. CPD10 could stabilize the GRK2 monomer (cyan, right) and prevent phospho-S670-GRK2 and TOMM6 aggregation (left); OMM: outer mitochondrial membrane; IMS: intermembrane space. Potential energies (E) of GRK2 (lower right) and phospho-S670-GRK2 (p-S670; lower left) without (w/o) and with CPD10 (+C10) interaction are given after energy minimization without (w/o MD sim) and with MD simulation (MD sim). See also and .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Affinity Chromatography, Western Blot, Control, Transgenic Assay, Two Tailed Test, Phospho-proteomics, Binding Assay, Inhibition, Membrane

GRK2 function modulation counteracts dysfunctional phospho-S670-GRK2 aggregation and retards AD progression and mortality in mice (A and B) Immunoblot of hippocampal, cytosolic GRK2 (A, left) and p-S670-GRK2 (A, right) of 18-month-old, untreated (18 mo), CPD10-treated (18mo + CPD10; 8 mg/kg/d), and 12-month-old, untreated (12 mo) Tg2576 mice. The control blot detects ADSL. Data are mean ± SD; n = 4 mice; one-way ANOVA and Tukey’s test; F(2,9) = 23.54, 37.45 (B). Below the right panel (A), brain and serum concentrations, and brain-to-serum ratios of CPD10 (mean ± SD; n = 6 mice) are shown. See also G and S5H. (C and D) Immunoblot of hippocampal, mitochondrial GRK2 (C) and TOMM6 (D) of 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) Tg2576 mice. The control blot detects TOMM40. Data represent mean ± SD; n = 4 biological replicates; unpaired, two-tailed t test; df = 6; t = 6.179, 1.729 (C); t = 7.540, 20.48 (D). (E and F) Hippocampal mitochondrial ACO2 activity without and with reactivation (E), and ATP contents (F) were determined in 18-month-old, untreated (18 mo), CPD10-treated (18mo + CPD10), and 12-month-old, untreated (12 mo) Tg2576 mice. Data represent mean ± SD ( n = 4 mice per group); one-way ANOVA and Tukey’s test; F(2,9) = 135.2, 13.11 (E); 30.06 (F). (G and H) Immunoblot of synaptosomal, hippocampal SNAP25 in 18-month-old, untreated (18 mo), CPD10-treated (18mo + CPD10), and 12-month-old, untreated (12 mo) Tg2576 mice. The control blot detects GNAO. Data are mean ± SD ( n = 4 mice per group); one-way ANOVA and Dunnett’s test; F(2,9) = 11.64 (G). (I) Immunofluorescence of mitochondrial TOMM40 (red) and GRK2 (green) in the hippocampal CA1 area of brain specimens from 18-month-old, CPD10-treated (Tg2576 18mo + CPD10) and untreated (Tg2576 18mo) Tg2576 mice. Nuclei were stained with DAPI (blue); scale bars: 20 μm. Immunofluorescence is representative of n = 7 mice per group. Three replicates are shown in . Four replicates are shown in A and S1D. (J) Overrepresentation analysis of significantly up-regulated transcripts (≥2-fold; p < 0.05) in frontal cortices of 18-month-old CPD10-treated Tg2576 mice compared to untreated mice. Biological processes (GO:BP) with p values <0.001 in the overrepresentation analysis and involvement in neurogenesis are shown. p values are presented as negative log10 of adjusted p values. Data are shown in . (K) Heatmap of significantly different ( p < 0.05) transcripts ( NeuN , Gfap , and Cx3cr1 ) in frontal cortices of 18-month-old, CPD10-treated Tg2576 (AD + C10) compared to untreated Tg2576 mice (AD). Data are shown in . (L) Activities of hippocampal α-, β-, and γ-secretase in 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) Tg2576 mice. Data represent mean ± SD ( n = 6 mice per group); unpaired, two-tailed t test; df = 10, t = 4.303, 5.280, 5.049. (M) Immunohistological detection of Aβ plaques (brown) on coronal brain sections of 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) female (f) and male (m) Tg2576 mice. Counterstaining was performed with hematoxylin (HE). Immunohistology is representative of six mice per group (scale bar: 200 μm). Replicates and quantitative data are shown in A and S5B. (N and O) Insoluble (N) and soluble (O) Aβ1-40 and Aβ1-42 in brains of 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) Tg2576 mice. Data represent mean ± SD ( n = 6 mice per group); unpaired, two-tailed t test; df = 10; t = 14.91, 16.01 (N); 5.352, 2.937 (O). (P) Probability of survival of CPD10-treated (+CPD10) and untreated Tg2576 mice ( n = 18 mice) was determined by Kaplan-Meier survival analysis with log rank (Mantel-Cox) test; df = 1, chi-square = 6.800. See also A, S1D–S1H, , , and ; and .

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: GRK2 function modulation counteracts dysfunctional phospho-S670-GRK2 aggregation and retards AD progression and mortality in mice (A and B) Immunoblot of hippocampal, cytosolic GRK2 (A, left) and p-S670-GRK2 (A, right) of 18-month-old, untreated (18 mo), CPD10-treated (18mo + CPD10; 8 mg/kg/d), and 12-month-old, untreated (12 mo) Tg2576 mice. The control blot detects ADSL. Data are mean ± SD; n = 4 mice; one-way ANOVA and Tukey’s test; F(2,9) = 23.54, 37.45 (B). Below the right panel (A), brain and serum concentrations, and brain-to-serum ratios of CPD10 (mean ± SD; n = 6 mice) are shown. See also G and S5H. (C and D) Immunoblot of hippocampal, mitochondrial GRK2 (C) and TOMM6 (D) of 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) Tg2576 mice. The control blot detects TOMM40. Data represent mean ± SD; n = 4 biological replicates; unpaired, two-tailed t test; df = 6; t = 6.179, 1.729 (C); t = 7.540, 20.48 (D). (E and F) Hippocampal mitochondrial ACO2 activity without and with reactivation (E), and ATP contents (F) were determined in 18-month-old, untreated (18 mo), CPD10-treated (18mo + CPD10), and 12-month-old, untreated (12 mo) Tg2576 mice. Data represent mean ± SD ( n = 4 mice per group); one-way ANOVA and Tukey’s test; F(2,9) = 135.2, 13.11 (E); 30.06 (F). (G and H) Immunoblot of synaptosomal, hippocampal SNAP25 in 18-month-old, untreated (18 mo), CPD10-treated (18mo + CPD10), and 12-month-old, untreated (12 mo) Tg2576 mice. The control blot detects GNAO. Data are mean ± SD ( n = 4 mice per group); one-way ANOVA and Dunnett’s test; F(2,9) = 11.64 (G). (I) Immunofluorescence of mitochondrial TOMM40 (red) and GRK2 (green) in the hippocampal CA1 area of brain specimens from 18-month-old, CPD10-treated (Tg2576 18mo + CPD10) and untreated (Tg2576 18mo) Tg2576 mice. Nuclei were stained with DAPI (blue); scale bars: 20 μm. Immunofluorescence is representative of n = 7 mice per group. Three replicates are shown in . Four replicates are shown in A and S1D. (J) Overrepresentation analysis of significantly up-regulated transcripts (≥2-fold; p < 0.05) in frontal cortices of 18-month-old CPD10-treated Tg2576 mice compared to untreated mice. Biological processes (GO:BP) with p values <0.001 in the overrepresentation analysis and involvement in neurogenesis are shown. p values are presented as negative log10 of adjusted p values. Data are shown in . (K) Heatmap of significantly different ( p < 0.05) transcripts ( NeuN , Gfap , and Cx3cr1 ) in frontal cortices of 18-month-old, CPD10-treated Tg2576 (AD + C10) compared to untreated Tg2576 mice (AD). Data are shown in . (L) Activities of hippocampal α-, β-, and γ-secretase in 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) Tg2576 mice. Data represent mean ± SD ( n = 6 mice per group); unpaired, two-tailed t test; df = 10, t = 4.303, 5.280, 5.049. (M) Immunohistological detection of Aβ plaques (brown) on coronal brain sections of 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) female (f) and male (m) Tg2576 mice. Counterstaining was performed with hematoxylin (HE). Immunohistology is representative of six mice per group (scale bar: 200 μm). Replicates and quantitative data are shown in A and S5B. (N and O) Insoluble (N) and soluble (O) Aβ1-40 and Aβ1-42 in brains of 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) Tg2576 mice. Data represent mean ± SD ( n = 6 mice per group); unpaired, two-tailed t test; df = 10; t = 14.91, 16.01 (N); 5.352, 2.937 (O). (P) Probability of survival of CPD10-treated (+CPD10) and untreated Tg2576 mice ( n = 18 mice) was determined by Kaplan-Meier survival analysis with log rank (Mantel-Cox) test; df = 1, chi-square = 6.800. See also A, S1D–S1H, , , and ; and .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Western Blot, Control, Two Tailed Test, Activity Assay, Immunofluorescence, Staining

A proteasome activity enhancer augments phospho-S670-GRK2 degradation and retards neurodegenerative AD symptoms (A) Binding energy (left), docking site (middle), and molecular docking of CPD57 to PSMA1 (chain E) and PSMA3 (chain F) of the 20S proteasome (8QYM) by CDOCKER. Data (left) are mean ± SD, of top 10 poses. (B) CPD57 ligand affinity chromatography (AP:C57) with brain protein lysates of 6-month-old B6 mice. CPD57-interacting PSMA3 (lanes 1 and 2) was detected in immunoblot (IB). Lanes 3 and 4 are controls from a control affinity matrix (AP: Con.). Two biological replicates are shown (1,3; 2,4). (C) Nano-LC-ESI-MS/MS identification of PSMA3 as a CPD57-interacting protein in brain lysates of B6 mice. (D) Brain contents of CPD57 (left) and brain-to-serum ratios (right) of 18-month-old Tg2576 mice after 6 months of treatment with the indicated doses of C57. Data are mean ± SD ( n = 6 mice per group); one-way ANOVA and Tukey’s test; F(3,20) = 16.93 (left); F(2,15) = 2.695 (right). (E) Enhanced proteasome activity in hippocampi of 18-month-old Tg2576 (AD) mice after 14 days of treatment with CPD57 (AD+57; 10 mg/kg/d). As indicated, the 20S proteasome was activated by SDS (0.03%, +). Proteasome activity is presented as % of the untreated AD control (mean ± SD; n = 4 mice per group); one-way ANOVA and Tukey’s test; F(3,12) = 38.09. (F) Immunoblot (IB) of PSMA3 in hippocampal, cytosolic proteins of 18-month-old Tg2576 mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). The right panel shows quantitative immunoblot data (df = 6; t = 12.14). (G and H) Immunoblot (IB) of phospho-S670-GRK2 (G, left) and GRK2 with GRK2-specific antibodies against the N-terminus of GRK2 (G, right) in hippocampal cytosolic proteins of 18-month-old Tg2576 AD mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). The lower control blot detects ADSL. Bar graphs (H) show quantitative data (df = 6; t = 14.45, 5.535). (I and J) Immunoblot (IB) of phospho-S670-GRK2 (I, left) and GRK2 with GRK2-specific antibodies against the N-terminus (I, right) on hippocampal mitochondria of 18-month-old Tg2576 mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). The control blot detects TOMM40. Bar graphs (J) show quantitative data (df = 6; t = 2.805, 2.858; 5.266, 12.40). (K) Immunoblot (IB) of TOMM6 on hippocampal mitochondria of 18-month-old Tg2576 mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). Bar graphs (right panels) show quantitative immunoblot data of aggregated (aggr.) and monomeric (mono.) TOMM6 (df = 6; t = 6.417, 9.231). (F, H, J, and K) Data represent mean ± SD; n = 4 mice per group (F and H) and n = 4 biological replicates per group (J and K); unpaired, two-tailed t test. (L) Immunohistological detection of hippocampal Aβ plaques (brown) on coronal brain sections of 18-month-old Tg2576 male (m) mice without treatment (AD) and with 6 months of CPD57 treatment (+57; 10 mg/kg/d). Nuclei were stained with hematoxylin (HE); scale bar: 200 μm. Immunohistology is representative of six mice per group. Replicates and quantitative data are shown in A and S5B. (M and N) Insoluble (M) and soluble (N) Aβ1-40 (upper) and Aβ1-42 (lower) in brains of 18-month-old Tg2576 mice without (AD) and with 6 months of CPD57 treatment (+57). Data are mean ± SD ( n = 6 mice per group); unpaired, two-tailed t test; df = 10; t = 12.73, 13.34 (M); 4.822, 3.117 (N). (O) Proteasome activities of brains from 18-month-old Tg2576 (AD) mice after 6 months of treatment with the indicated doses of CPD57. (P) Contents of insoluble Aβ1-40 in brains of 18-month-old Tg2576 (AD) mice after 6 months of treatment with the indicated doses of CPD57. (O and P) Data are mean ± SD ( n = 6 mice per group); one-way ANOVA and Tukey’s test; F(3,20) = 34.26 (Suc-LLVY-AMC), 32.49 (Boc-LRR-AMC), 27.87 (Z-LLE-AMC), 40.90 (P). See also .

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: A proteasome activity enhancer augments phospho-S670-GRK2 degradation and retards neurodegenerative AD symptoms (A) Binding energy (left), docking site (middle), and molecular docking of CPD57 to PSMA1 (chain E) and PSMA3 (chain F) of the 20S proteasome (8QYM) by CDOCKER. Data (left) are mean ± SD, of top 10 poses. (B) CPD57 ligand affinity chromatography (AP:C57) with brain protein lysates of 6-month-old B6 mice. CPD57-interacting PSMA3 (lanes 1 and 2) was detected in immunoblot (IB). Lanes 3 and 4 are controls from a control affinity matrix (AP: Con.). Two biological replicates are shown (1,3; 2,4). (C) Nano-LC-ESI-MS/MS identification of PSMA3 as a CPD57-interacting protein in brain lysates of B6 mice. (D) Brain contents of CPD57 (left) and brain-to-serum ratios (right) of 18-month-old Tg2576 mice after 6 months of treatment with the indicated doses of C57. Data are mean ± SD ( n = 6 mice per group); one-way ANOVA and Tukey’s test; F(3,20) = 16.93 (left); F(2,15) = 2.695 (right). (E) Enhanced proteasome activity in hippocampi of 18-month-old Tg2576 (AD) mice after 14 days of treatment with CPD57 (AD+57; 10 mg/kg/d). As indicated, the 20S proteasome was activated by SDS (0.03%, +). Proteasome activity is presented as % of the untreated AD control (mean ± SD; n = 4 mice per group); one-way ANOVA and Tukey’s test; F(3,12) = 38.09. (F) Immunoblot (IB) of PSMA3 in hippocampal, cytosolic proteins of 18-month-old Tg2576 mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). The right panel shows quantitative immunoblot data (df = 6; t = 12.14). (G and H) Immunoblot (IB) of phospho-S670-GRK2 (G, left) and GRK2 with GRK2-specific antibodies against the N-terminus of GRK2 (G, right) in hippocampal cytosolic proteins of 18-month-old Tg2576 AD mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). The lower control blot detects ADSL. Bar graphs (H) show quantitative data (df = 6; t = 14.45, 5.535). (I and J) Immunoblot (IB) of phospho-S670-GRK2 (I, left) and GRK2 with GRK2-specific antibodies against the N-terminus (I, right) on hippocampal mitochondria of 18-month-old Tg2576 mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). The control blot detects TOMM40. Bar graphs (J) show quantitative data (df = 6; t = 2.805, 2.858; 5.266, 12.40). (K) Immunoblot (IB) of TOMM6 on hippocampal mitochondria of 18-month-old Tg2576 mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). Bar graphs (right panels) show quantitative immunoblot data of aggregated (aggr.) and monomeric (mono.) TOMM6 (df = 6; t = 6.417, 9.231). (F, H, J, and K) Data represent mean ± SD; n = 4 mice per group (F and H) and n = 4 biological replicates per group (J and K); unpaired, two-tailed t test. (L) Immunohistological detection of hippocampal Aβ plaques (brown) on coronal brain sections of 18-month-old Tg2576 male (m) mice without treatment (AD) and with 6 months of CPD57 treatment (+57; 10 mg/kg/d). Nuclei were stained with hematoxylin (HE); scale bar: 200 μm. Immunohistology is representative of six mice per group. Replicates and quantitative data are shown in A and S5B. (M and N) Insoluble (M) and soluble (N) Aβ1-40 (upper) and Aβ1-42 (lower) in brains of 18-month-old Tg2576 mice without (AD) and with 6 months of CPD57 treatment (+57). Data are mean ± SD ( n = 6 mice per group); unpaired, two-tailed t test; df = 10; t = 12.73, 13.34 (M); 4.822, 3.117 (N). (O) Proteasome activities of brains from 18-month-old Tg2576 (AD) mice after 6 months of treatment with the indicated doses of CPD57. (P) Contents of insoluble Aβ1-40 in brains of 18-month-old Tg2576 (AD) mice after 6 months of treatment with the indicated doses of CPD57. (O and P) Data are mean ± SD ( n = 6 mice per group); one-way ANOVA and Tukey’s test; F(3,20) = 34.26 (Suc-LLVY-AMC), 32.49 (Boc-LRR-AMC), 27.87 (Z-LLE-AMC), 40.90 (P). See also .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Activity Assay, Binding Assay, Affinity Chromatography, Western Blot, Control, Tandem Mass Spectroscopy, Two Tailed Test, Staining

Journal: Cell Reports Medicine

Article Title: Therapeutic base editing alleviates restrictive cardiomyopathy

doi: 10.1016/j.xcrm.2026.102639

Figure Lengend Snippet: Intrathoracic ABE treatment corrects Tnni3 R193H mutation in cardiac tissue (A and B) Schematic illustration of the dual AAV9 vector construction strategy and ABE treatment, administered via intrathoracic injections in 6-week-old Tnni3 R193H/R193H mice. (C and D) Efficient correction of Tnni3 mutation in the heart. Assessment of gene editing efficiency in the hearts of Tnni3 R193H/R193H mice 12 weeks after the AAV treatment. A>G editing efficiencies were measured in genomic DNA and mRNA using high-throughput sequencing. Each point represents an individual mouse. ∗∗∗∗ p < 0.0001 based on Student’s unpaired t test analysis. Data are presented as mean ± SEM. (E and F) Lack of liver editing following the AAV9-ABE treatment. As in (C), gene editing efficiency in the livers of Tnni3 R193H/R193H mice after 12 weeks of dual AAV treatment. A>G editing efficiency was also assessed in the genomic DNA and mRNA extracted from liver tissue, demonstrating diminished delivery and expression of the ABE system in the hepatic tissue. Each point represents an individual mouse. ns, not statistically significant (Student’s unpaired t test analysis). Data are presented as mean ± SEM.

Article Snippet: The plasmids Cbh_v5 AAV9-ABE N-terminus (Addgene plasmid no. 137177) and Cbh_v5 AAV9-ABE C-terminus (Addgene plasmid no. 137178) were employed for AAV plasmid construction.

Techniques: Mutagenesis, Plasmid Preparation, Next-Generation Sequencing, Expressing

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