ldb3  (Proteintech)

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: (A) WT and A165V mutant Ldb3 mRNA sequences are shown, highlighting the mutation site (red). Nineteen candidate siRNAs were designed to target this region; the five selected sequences (sense strand), chosen based on computational predictions and prior evidence, are indicated by arrows. siRNAs were numbered according to their position relative to the mismatch with the WT sequence and evaluated in HEK-293 cells expressing either WT or mutant LDB3. (B and C) Immunoblot analysis performed 48 h post-transfection showing effect of the siRNAs on total LDB3 protein levels in mutant- and WT-LDB3 expressing cells (n = 3 per group), including si-SC and NT controls. (D and E) AS-qPCR quantification of mutant and WT Ldb3 transcripts (n = 3 per group). Transcript levels were normalized to Gapdh using the ΔΔCt method and immunoblots were normalized to GAPDH. (F) Schematic of the miR30-based shRNA construct encoding either si10-Ldb3-A165V or si16-Ldb3-A165V (top). The construct was packaged into AAV9 for intramuscular delivery. Following cellular entry, shRNAmiR transcripts are processed through the endogenous microRNA pathway to generate mature shRNAs targeting mutant Ldb3 mRNA (bottom). (G) Schematic of the dose-optimization strategy for allele-specific Ldb3 targeting. Three-month-old Ldb3 Ala165Val/+ mice received a single intramuscular injection of AAV-shRNAmiR (sh10 or sh16), and TA muscles were analyzed 1 month later. (H) Schematic of the therapeutic disease-reversal paradigm. Ldb3 Ala165Val/+ mice were injected intramuscularly with AAV-shRNAmiR (sh10 or sh16) at 5 months of age, and outcomes were evaluated at 1 and 3 months post-injection. All data are presented as mean ± SD of three biological replicates (n = 3) with technical triplicates. Only statistically significant comparisons are shown. *P < 0.05, **P < 0.01 and ***P < 0.001, as determined by Welch’s two-sample t test. si-SC, scrambled siRNA control; NT, no-treatment control. F, G and H created from Biorender.com.

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Mutagenesis, Sequencing, Expressing, Western Blot, Transfection, shRNA, Construct, Injection, Muscles, Control

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: 5-month-old Ldb3 Ala165Val/+ mice received a single intramuscular injection of sh10 or sh16 into the TA muscle; contralateral TA muscles received scramble control (SC). (A and B) AS-qPCR analysis showing relative expression of WT (A165A) and mutant Ldb3 -A165V transcripts at 1 and 3 months post-injection compared to SC-treated controls in sh16 and sh10 treatment groups. (C and D) Representative immunoblots and densitometric quantification showing total LDB3 protein levels in the sh16 and sh10 treated muscles at 1 and 3 months post-injection compared to SC-treated controls. AS-qPCR and immunoblot data were obtained from 3-7 animals per group and triplicate assays. AS-qPCR data were normalized to Myom1 using the ΔΔCt method, and immunoblots were normalized to endogenous vinculin protein. (E and F) Adjacent transverse cryosections of frozen TA muscles harvested 3 months posttreatment was stained with modified Gomori trichrome (MGT), NADH-TR, and filamin C. Representative images show sarcoplasmic aggregates and abnormal oxidative enzyme activity (black arrowheads) in SC-treated controls (top panels) compared with sh16- and sh10-treated muscles (bottom panels). Filamin C-positive aggregates are indicated by white arrowheads in SC-treated and absence in sh16 and sh10 treated muscle. (G and H) Representative images of frozen transverse TA muscle cryosections stained with wheat germ agglutinin (WGA; cyan) showing internal nuclei (red dots in magnified insets), together with scatter bar plots quantifying the percentage of fibers containing internal nuclei in sh16- and sh10-treated muscles compared with SC-treated controls. Scale bars: MGT, NADH-TR, and filamin C: 50 µm; WGA: 250 µm. (N = 3-6 TA muscles per group). (I and J) In-vivo assessment of muscle contractility in 5-month-old Ldb3 Ala165Val/+ mice treated with sh10 for 1 month. (I) Force-frequency relationship curves showing specific isometric torque generated across increasing stimulation frequencies in WT mice, sh10-treated TA muscles, and contralateral SC-treated TA muscles. (J) Quantification of peak specific isometric torque in WT, sh10-treated TA muscles, and contralateral SC-treated TA muscles. In the force-frequency curves, matching colored rectangles and circles denote shRNA-treated TA muscles and the corresponding contralateral SC-treated TA muscles from the same mice, respectively, while purple triangles denote PBS-treated WT TA muscles used as reference controls (n = 3-4 mice per group). Data are presented as mean ± SD (A-F) and mean ± SEM (J). Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant, as determined by an unpaired, two-tailed Student’s t-test (A, B, I and J) and Welch’s t-test (C and D).

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Injection, Muscles, Control, Expressing, Mutagenesis, Western Blot, Staining, Modification, Activity Assay, In Vivo, Generated, shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: Representative IF images of LDB3 and filamin C in PFA perfused longitudinal TA muscle sections from advanced-stage, (5-month-old) Ldb3 Ala165Val/+ mice treated with sh-SC, sh16, or sh10 ( A and B) for one month (upper panel) or three months (bottom panel) (n = 4-6 per group). (C and D) Quantification of muscle fibers containing sarcoplasmic filamin C aggregates in sh-SC control and shRNA-treated groups. Bar scatter plots represent the percentage of fibers exhibiting filamin C aggregates, quantified from at least five randomly selected areas (> 50 fibers per muscle) from immunostained TA sections (n = 3 per group). (E and F) Representative IF images of BAG3 and HSPA8 in longitudinal TA cryosections from advanced-stage, (5-month-old) Ldb3 Ala165Val/+ mice treated with sh-SC, sh16, or sh10 for 1 month (upper panels) or 3 months (lower panels) (n = 4-6 per group). Data are expressed as mean ± SEM, with statistical significance denoted by ****P < 0.0001, determined using an unpaired, two-tailed Student’s t-test. Scale bars represent 50 µm (A and B) and 20 µm (E and F).

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Control, shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: Representative immunoblots showing PKCα protein levels in frozen TA muscle from 5-monthold Ldb3 Ala165Val/+ mice treated with AAV9-shRNAmiR constructs and age-matched PBS treated WT controls. (A and B) PKCα protein levels following sh16 (A) or sh10 (B) treatment at 1 and 3 months post-injection. Bar-scatter plots below the blots show densitometric quantification of PKCα normalized to endogenous vinculin (n = 3 per group and triplicate assays). Ldb3 Ala165Val/+ mice were administered either scramble control (SC) or shRNAmiR targeting sh16 or sh10. Data are presented as mean ± SD, with statistical significance levels of **P < 0.01, ***P < 0.001, ****P < 0.0001, calculated using a two-tailed unpaired Student’s t-test.

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Western Blot, Construct, Injection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: Three-month-old Ldb3 Ala165Val/+ mice received a single intramuscular injection of sh10 or sh16 into the TA muscle; contralateral TA muscles received scramble control (SC). (A) Schematic of the early-intervention (disease-prevention) paradigm. Ldb3 Ala165Val/+ mice were injected intramuscularly with AAV-shRNAmiR (sh10 or sh16) at 3 months of age, and outcomes were evaluated at 1 and 3 months post-injection (created from Biorender.com ). (B and C) AS-qPCR analysis showing relative expression of WT (A165A) and mutant Ldb3 -A165V transcripts at 1 and 3 months post-injection compared to scramble-treated controls (SC) in sh16 and sh10 treatment groups. (D and E) Representative immunoblots and densitometric quantification showing total LDB3 protein levels in the sh16 and sh10 treated muscles at 1 and 3 months post-injection compared to SC-treated controls. AS-qPCR and immunoblot data were obtained from 3-6 animals per group and triplicate assays. AS-qPCR data were normalized to endogenous Myom1 using the ΔΔCt method, and immunoblots were normalized to endogenous vinculin protein. Data are presented as mean ± SD, with statistical significance defined as **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant, as determined by unpaired, two-tailed Student’s t-test (B and C) and Welch’s t test (D and E).

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Injection, Muscles, Control, Expressing, Mutagenesis, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: (A and C) Representative IF images of LDB3 and filamin C in PFA perfused longitudinal TA muscle sections from early-stage (3-month-old) Ldb3 Ala165Val/+ mice treated with sh-SC, sh16 (A), or sh10 (C) for one month (upper panel) or three months (lower panel) (n = 4-6 per group). (B and D) Quantification of muscle fibers containing sarcoplasmic filamin C aggregates in sh-SC and shRNA-treated groups corresponding to panels (A) and (C). Bar-scatter plots represent the percentage of fibers exhibiting filamin C aggregates, quantified from at least five randomly selected fields (>50 fibers per muscle) per section (n = 3 per group). (E and F) Representative IF images of BAG3 and HSPA8 in longitudinal TA cryosections from early-stage, (3-month-old) Ldb3 Ala165Val/+ mice treated with sh-SC, sh16, or sh10 for 1 month (upper panels) or 3 months (lower panels) (n = 4-6 per group). Data are expressed as mean ± SEM, with statistical significance denoted by **P < 0.01, determined using an unpaired, two-tailed Student’s t-test. Scale bars represent 50 µm (A and C) and 20 µm (E and F).

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: Representative immunoblots showing PKCα protein levels in frozen TA muscle from 3-monthold Ldb3 Ala165Val/+ mice treated with AAV9-shRNAmiR constructs and age-matched PBS treated WT controls. (A and B) PKCα expression following sh16 (A) or sh10 (B) treatment at 1 and 3 months post-injection. Bar-scatter plots below the blots show densitometric quantification of PKCα normalized to endogenous vinculin (n = 3 per group and triplicate assays). Ldb3 Ala165Val/+ mice were administered either scramble control (SC) or shRNAmiR targeting sh16 or sh10. Data are presented as mean ± SD, with statistical significance levels of *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, calculated using a two-tailed unpaired Student’s t-test. Phosphoproteomic profiling was performed to compare shRNA-treated and SC-treated TA muscles (n = 3 per group). (C) Volcano plot of differentially phosphorylated proteins in sh16-treated versus SC-treated TA muscle. Each point represents a phosphoprotein; red points indicate significantly upregulated phosphosites and green points indicate significantly downregulated phosphosites (adjusted P < 0.05), whereas gray points denote non-significant changes. The x axis represents log□ fold change (treatment/control), and the y axis represents −log□□ adjusted P values. A total of 35 phosphoproteins met significance criteria (adjusted P < 0.05). (D) Pathway and process enrichment analysis of differentially phosphorylated proteins. Significantly enriched MGI Phenotype and Gene Ontology (GO) Biological Process terms are shown. Bars represent−log□□ (P value); unadjusted P values are displayed. (E) Protein-protein interaction network constructed from significantly altered phosphoproteins. Nodes represent phosphoproteins; edges indicate curated interactions. PKCα is positioned within the network to visualize connectivity among structural and signaling modules. (F) KEA3 kinase enrichment analysis. Mean rank heat bar showing top-ranked upstream kinases inferred from the phosphoproteomic dataset across multiple kinase-substrate libraries (PhosD, STRING, and Kinase Library). Color scale reflects relative ranking within each database. (G) Cheng-KSIN kinase enrichment analysis. Bar plot and corresponding network visualization of kinases enriched based on motif-derived kinase–substrate interactions. Enrichment is represented as −log(P value). (H) PhosD all-kinase ranking. Top kinases ranked using kinase-substrate co-phosphorylation associations derived from the PhosD library. (I) PhosD kinase interaction mini-network illustrating inferred relationships among PRKCA (PKCα) and associated signaling nodes.

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Western Blot, Construct, Expressing, Injection, Control, Two Tailed Test, shRNA, Muscles, Derivative Assay, Phospho-proteomics

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: Weighted gene coexpression network analysis of the GSE153434 dataset and Venn diagram to obtain two crucial genes. (A) A soft threshold power value of 8 was used, which is the optimum option. (B) Demonstration of scale‐free network validation with a soft threshold of 8. (C) By clustering genes with strong correlations into the same module, different modules were generated. Different modules are displayed in different colors. (D) Network heatmap showing branching of overall genes associated with modules in a hierarchical clustering dendrogram. (E) Analysis of the correlation between each module and TAAD. (F) The black module was significantly positively correlated with TAAD (correlation coefficient = 0.94, p < 0.001). (G) Venn diagram showing the intersection of DEGs obtained after RRA analysis, the WGCNA hub genes, and the TAAD‐related genes in GeneCards, finally yielding TIMP1 and LDB3 as the crucial genes.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Biomarker Discovery, Generated

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: Single‐cell sequencing analysis in the GSE213740 dataset. (A) Cell sample quality was assured by the analysis of three metrics: RNA count, gene count, and mitochondrial gene percentage. (B) The 2000 most highly variable genes are shown in red, with the top 10 highly variable genes marked. (C) Dimensionality reduction and clustering analysis were performed on the cells in the dataset, and the clustering of the TAAD and control groups is displayed in the ‘tSNE’ diagram. (D) Annotation of the clustered cells, including T cells, smooth muscle cells, fibroblasts, macrophages, mesenchymal cells, endothelial cells, monocytes, plasmocytes, giant cells, and B cells. (E) Expression of TIMP1 and LDB3 in cell clusters.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Sequencing, Control, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: Pseudochronological analysis and pathway enrichment analysis of smooth muscle cells in the GSE213740 dataset. (A) Smooth muscle cells in the dataset were extracted and reclustered into nine clusters. (B) Expression analysis of LDB3 in smooth muscle cells from the GSE213740 dataset. (C) Pseudotime analysis of smooth muscle cells. The three images in the illustration show the three different differentiation states based on the aggregation of nine cell clusters and the differences in the time series of cell differentiation. (D) GO enrichment analysis of significant DEGs between cell clusters 1 and 5 (with considerable LDB3 expression) and the remaining seven cell clusters (with insignificant LDB3 expression). (E) KEGG enrichment analysis based on significantly differentially expressed genes between cell clusters 1 and 5 and the remaining seven cell clusters.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Expressing, Cell Differentiation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: LDB3 SNPs analysis in the UK Biobank. Univariate (19 SNPs) and multivariate (rs34346901 and rs117443987) associations between SNPs and aortic aneurysm and dissection. Six SNPs were significantly related, and the relative ratios of each genotype for aortic aneurysm and dissection are shown in the pie charts.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Dissection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: Validation of LDB3 in human samples and upregulation of LDB3 by Ang II stimulation in HA‐VSMC. (A) Histology staining of TAAD and normal tissue. HE, Masson's trichrome staining, and EVG staining were used to reveal the differences in cellularity, collagen fibers, and elastic fibers in ascending aorta sections of TAAD and normal tissues. (B) Tissue collagen and elastin quantification ( n = 10). (C) Immunofluorescence staining showed that the expression of LDB3 was significantly decreased in TAAD vascular smooth muscle cells. (D, E) Long and short isoforms of LDB3 were expressed in TAAD aortic wall samples, and Western blot verification revealed that both long and short LDB3 expressions were significantly downregulated in TAAD compared to normal samples ( n = 10). (F, G) Ang II induced HA‐VSMC significant bands of LDB3 and quantification analysis. (H, I) Immunofluorescence staining of LDB3 and phalloidin in HA‐VSMC after Ang II treatment and quantification analysis ( n = 4). * p < 0.05, ** p < 0.01, **** p < 0.0001 in comparisons of two groups as indicated or compared with the corresponding control.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Biomarker Discovery, Staining, Immunofluorescence, Expressing, Western Blot, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of the Hub Gene LDB3 in Stanford Type A Aortic Dissection Based on Comprehensive Bioinformatics Analysis

doi: 10.1111/jcmm.70471

Figure Lengend Snippet: Validation of LDB3 expression in Ang II‐induced aortic dissection aneurysm models. (A) Representative photographs showing macroscopic features of dissection aneurysms induced by Ang II. (B, C) Representative ultrasound images of aortic dimension in two groups and quantification analysis ( n = 5). (D, E) HE, Masson's trichrome staining, and EVG staining and elastin degradation score analysis ( n = 5). (F, G) Immunofluorescence staining of LDB3 in Ang II‐induced aortic dissection aneurysm and its quantification analysis ( n = 5). * p < 0.05, ** p < 0.01, **** p < 0.0001 in comparisons of two groups as indicated or compared to the corresponding control.

Article Snippet: Antibodies against α‐SMA (14395‐1‐AP; Proteintech, 1:100) and LDB3 (11004‐1‐AP; Proteintech, 1:100) were then used for staining.

Techniques: Biomarker Discovery, Expressing, Dissection, Staining, Immunofluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Association between ZASP/LDB3 Pro26Ser and Inclusion Body Myopathy

doi: 10.3390/ijms25126547

Figure Lengend Snippet: Family pedigree with black symbols indicating the affected members. The arrow indicates the proband ( A ). Electropherograms in the proband, his sister, and their father ( B ). Representative Western blot of controls and patient samples. Lane 1–4 staining with LDB3 (78 KDa and 32 KDa) and desmin. Lane 5–8 staining with LDB3 and myotilin. α-actinin-1 was used as loading control ( C ). Schematic representation of LDB3 protein structure with the indication of the already reported variants. The arrow shows the localization of our patient’s mutation in the PDZ domain ( D ).

Article Snippet: Membranes were probed with antibodies to LDB3 (1:15,000, goat polyclonal; ab110003 Abcam, Cambridge, UK), to myotilin (1:250, mouse monoclonal; Novocastra, Newcastle upon Tyne, UK), to desmin (1:200, mouse monoclonal; Novocastra, Newcastle upon Tyne, UK), and α-actinin-1 (1:5000; Sigma-Aldrich, Burlington, MA, USA). α-actinin-1 was used as an indicator of protein loading.

Techniques: Western Blot, Staining, Mutagenesis