Journal: The Journal of Pathology: Clinical Research

Article Title: A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation‐prone Ano5 peptide

doi: 10.1002/cjp2.92

Figure Lengend Snippet: Screening of the anti‐Ano5 antibodies by western blotting analysis. AD293 cells infected with adenoviruses expressing GFP or FLAG‐hANO5‐YFP were analysed by western blotting with anti‐GFP (A), anti‐FLAG (B), Santa Cruz T‐12 anti‐Ano5 (C), UC Davis/NIH NeuroMab Facility anti‐Ano5 (D), or GAPDH (bottom panel in A and B). The molecular mass standards (Precision Plus Standard; Bio‐Rad) are shown in kilodalton on the right for each panel. All experiments were repeated at least three times.

Article Snippet: Mouse Ano5‐mCherry or mCherry‐C1 cassettes were subcloned into pLVX‐mCherry‐C1 vector (Clontech) for making Ano5 lentiviruses.

Techniques: Western Blot, Infection, Expressing

Journal: The Journal of Pathology: Clinical Research

Article Title: A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation‐prone Ano5 peptide

doi: 10.1002/cjp2.92

Figure Lengend Snippet: Confocal imaging of recombinant Ano5 immunostained using N421A/85 Ab. (A) AD293 cells infected with adenoviruses expressing GFP or FLAG‐hANO5‐YFP were stained with anti‐Ano5 antibody (N421A/85, red) and DAPI (blue). The green signals represent the expression of GFP or FLAG‐hANO5‐YFP. (B) AD293 cells infected with adenoviruses expressing GFP or FLAG‐hANO5‐YFP were stained with anti‐calnexin (an ER marker) antibody (red) and DAPI (blue). GFP or FLAG‐hANO5‐YFP is shown in green. Scale bar = 10 µm. These are representative of a minimum of three experiments.

Article Snippet: Mouse Ano5‐mCherry or mCherry‐C1 cassettes were subcloned into pLVX‐mCherry‐C1 vector (Clontech) for making Ano5 lentiviruses.

Techniques: Imaging, Recombinant, Infection, Expressing, Staining, Marker

Journal: The Journal of Pathology: Clinical Research

Article Title: A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation‐prone Ano5 peptide

doi: 10.1002/cjp2.92

Figure Lengend Snippet: Epitope mapping of the UC Davis anti‐Ano5 mAb (N421A/85). (A) Schematic showing different truncated constructs of human Ano5. The numbers in the boxes represent the positions of the amino acids of Ano5. Truncated and full‐length constructs expressing recombinant human Ano5 tagged with FLAG and GFP were analysed by western blotting with anti‐GFP (B), anti‐FLAG (C), or UC Davis anti‐Ano5 (N421A/85)(D) antibodies. The loading control of GAPDH for each panel is the same as in panel C. The molecular mass standards (Precision Plus Standard; Bio‐Rad) are shown in kilodalton on the right for each panel.

Article Snippet: Mouse Ano5‐mCherry or mCherry‐C1 cassettes were subcloned into pLVX‐mCherry‐C1 vector (Clontech) for making Ano5 lentiviruses.

Techniques: Construct, Expressing, Recombinant, Western Blot, Control

Journal: The Journal of Pathology: Clinical Research

Article Title: A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation‐prone Ano5 peptide

doi: 10.1002/cjp2.92

Figure Lengend Snippet: Expression of Ano5 in the skeletal muscle biopsies of human patients. (A) H&E staining of human skeletal muscle sections from one control (C1) and three ANO5 patients (P1–P3). (B) The human skeletal muscle lysates were analysed by SDS‐PAGE and immunoblotted with antibodies against Ano5 (B, N421A/85) or dystrophin (C). GAPDH was used as the loading control.

Article Snippet: Mouse Ano5‐mCherry or mCherry‐C1 cassettes were subcloned into pLVX‐mCherry‐C1 vector (Clontech) for making Ano5 lentiviruses.

Techniques: Expressing, Staining, Control, SDS Page

Journal: The Journal of Pathology: Clinical Research

Article Title: A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation‐prone Ano5 peptide

doi: 10.1002/cjp2.92

Figure Lengend Snippet: Immunofluorescence analysis of Ano5 in skeletal muscle cryosections from control (C1–3) and ANO5 (P1, P2) human patients. The human skeletal muscle cryosections were double‐stained with antibodies against Ano5 (green, N421A/85) and dystrophin (red). Nuclei were counterstained with DAPI (blue). Scale bar = 100 µm.

Article Snippet: Mouse Ano5‐mCherry or mCherry‐C1 cassettes were subcloned into pLVX‐mCherry‐C1 vector (Clontech) for making Ano5 lentiviruses.

Techniques: Immunofluorescence, Control, Staining

Journal: The Journal of Pathology: Clinical Research

Article Title: A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation‐prone Ano5 peptide

doi: 10.1002/cjp2.92

Figure Lengend Snippet: Aberrant splicing and production of truncated Ano5 peptide caused by c.363 + 4A > G mutation. (A) Diagram showing the ANO5 constructs carrying intron 6 (i6, WT, or mutant) between exons 6 and 7. The WT intron results in correct splicing while the mutant intron is expected to disrupt the splicing site and use a downstream site so that a 158‐bp sequence from intron 6 will be included in the cDNA. Red asterisk: mutant transcript; arrow: WT transcript. (B) RT‐PCR analysis of Ano5 expression in cells transfected with different constructs. (C, D) Immunoblotting of cells transfected with the Ano5 constructs using mAb‐Ano5 (C), anti‐FLAG (D), and anti‐GAPDH. Higher exposure of the upper region of the mutant lane in panel C is shown to the right of the panel. Red asterisk: mutant Ano5 peptide.

Article Snippet: Mouse Ano5‐mCherry or mCherry‐C1 cassettes were subcloned into pLVX‐mCherry‐C1 vector (Clontech) for making Ano5 lentiviruses.

Techniques: Mutagenesis, Construct, Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Western Blot

Journal: The Journal of Pathology: Clinical Research

Article Title: A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation‐prone Ano5 peptide

doi: 10.1002/cjp2.92

Figure Lengend Snippet: Clinical and genetic data of the control (C1–3) and ANO5 (P1–3) patients

Article Snippet: Mouse Ano5‐mCherry or mCherry‐C1 cassettes were subcloned into pLVX‐mCherry‐C1 vector (Clontech) for making Ano5 lentiviruses.

Techniques: Control

Journal: bioRxiv

Article Title: Dysregulation of a novel autophagosome-mitochondria contact contributes to autophagy dysfunction and neurodegeneration in tauopathy

doi: 10.64898/2026.03.23.713823

Figure Lengend Snippet: AV-Mito untethering relies on AV-bound TBC1D15 GAP activity and is required for autophagic cargo clearance in tauopathy axons. ( A and B ) Representative images ( A ) and quantitative analysis ( B ) of TBC1D15 localization in normal axons. Data were expressed as the percentage of AVs or mitochondria positive for TBC1D15 and quantified from the total numbers of AVs or mitochondria ( n , indicated in parentheses) from the axons of 51 neurons ( B ). Arrows: TBC1D15-positive AVs; arrowheads: a TBC1D15-negative AV in an AV-Mito contact; asterisk: mitophagosome. ( C and D ) Representative kymographs ( C ) and quantitative analysis ( D ) of TBC1D15-tagged AVs in axons with 24-hour trehalose incubation. The relative motility of AVs was quantified from the axons of 17 neurons as indicated in parentheses ( D ). Arrows: TBC1D15-tagged motile AVs. ( E - H ) Representative kymographs ( E ) and quantitative analysis ( F - H ) of AV-Mito contacts in axons expressing TBC1D15 or TBC1D15 D397A mutant. The data were quantified and expressed as AV-Mito contact duration and its frequency, the contact number per 100 μm axonal length, the percentage of AVs in contacts, and the relative motility of AVs. Data were quantified from the total numbers of neurons ( n ) and AV-Mito contacts (c) as indicated in parentheses ( F - H ). Arrows: AV-Mito contacts. ( I and J ) Representative images ( I ) and quantitative analysis ( J ) of p62 in tauP301S axons with and without OE of TBC1D15 or TBC1D15 D397A mutant. The data were quantified and expressed as GFP-p62 puncta number per 100 μm axonal length. Data were quantified from the total numbers of neurons ( n ) as indicated in parentheses ( J ). ( K and L ) Representative blots ( K ) and quantitative analysis ( L ) of tauP301S Tg neurons transduced with and without AAV-hTBC1D15. Protein intensities were normalized to those in control tauP301S neurons without TBC1D15 OE. Data were collected from two ( B and D ), three ( F-H and J ), or five ( L ) dissections. Data were expressed as the mean ± SEM and analyzed using linear mixed-effects models ( B , F - H , and J ) or by two-sided Student’s t -test ( L ). Scale bars: 10 μm.

Article Snippet: The AAV2/9-mCherry, AAV2/9-mCherry-hTBC1D15, and AAV2/9-mCherry-hTBC1D15 D397A mutant viruses were produced by Vector BioLabs.

Techniques: Activity Assay, Incubation, Expressing, Mutagenesis, Transduction, Control

Journal: bioRxiv

Article Title: Dysregulation of a novel autophagosome-mitochondria contact contributes to autophagy dysfunction and neurodegeneration in tauopathy

doi: 10.64898/2026.03.23.713823

Figure Lengend Snippet: TBC1D15 OE reduces AV-Mito hyper-tethering and alleviates p62 accumulation and tau pathology in tauopathy mouse brains. ( A and B ) Representative TEM images ( A ) and quantitative analysis ( B ) of the hippocampi in 8-month-old tauP301S Tg mouse brains infected with AAV-mCherry or AAV-mCherry-hTBC1D15. The number of presynaptic AVs, the percentage of presynaptic terminals containing AVs, and the number of AVs or mitochondria in AV-Mito contacts were quantified and normalized to or compared to those in control tauP301S Tg mouse brains. Data were collected from two mice per group; the total numbers of presynaptic terminals ( n ) are indicated in parentheses ( B ). ( C and D ) Representative images ( C ) and quantitative analysis ( D ) of p62 accumulation in 10-month-old tauP301S Tg mouse hippocampi with and without OE of TBC1D15 or TBC1D15 D397A. p62 fluorescence mean intensity in the hippocampal mossy fibers was quantified and normalized to that of AAV-mCherry-injected tauP301S Tg controls. Data were collected from three mice per group; the total numbers of imaging slice sections ( n ) are indicated in parentheses ( D ). SYP: synaptophysin. Arrows: p62 clusters at SYP-indicated presynaptic terminals. ( E and F ) Representative images ( E ) and quantitative analysis ( F ) of tau accumulation in 10-month-old non-Tg and tauP301S Tg hippocampi with and without OE of TBC1D15 or TBC1D15 D397A. AT8 and PHF1 antibody-marked phospho-tau mean intensities in the hippocampal mossy fibers were quantified and normalized to those of AAV-mCherry-injected tauP301S Tg control mice. Data were collected from three mice per group; the total numbers of imaging slice sections ( n ) are indicated in parentheses ( F ). ( G and H ) Representative blots ( G ) and quantitative analysis ( H ) of AT8 and PHF1 antibody-marked phospho-tau and Tau5 antibody-indicated total tau in sarkosyl-extractable and sarkosyl-insoluble fractions from 10-month-old tauP301S Tg mouse brains with and without TBC1D15 OE. Protein intensities were normalized to those in AAV-mCherry-injected tauP301S Tg controls. Data were quantified from four mice per group. Data were expressed as the mean ± SEM and analyzed using linear mixed-effects models ( B, D and F ) or by two-sided Student’s t -test ( H ). Scale bars: 100 nm ( A ), 10 μm ( C ), and 20 μm ( E ).

Article Snippet: The AAV2/9-mCherry, AAV2/9-mCherry-hTBC1D15, and AAV2/9-mCherry-hTBC1D15 D397A mutant viruses were produced by Vector BioLabs.

Techniques: Infection, Control, Fluorescence, Injection, Imaging

Journal: bioRxiv

Article Title: Dysregulation of a novel autophagosome-mitochondria contact contributes to autophagy dysfunction and neurodegeneration in tauopathy

doi: 10.64898/2026.03.23.713823

Figure Lengend Snippet: Attenuation of neurodegeneration and memory deficits in tauopathy mice with TBC1D15 OE. (A and B ) Representative images ( A ) and quantitative analysis ( B ) of presynaptic terminal and neuron densities in 10-month-old non-Tg and tauP301S Tg hippocampi with and without OE of TBC1D15 or TBC1D15 D397A. The mean intensity of SYP-indicated presynaptic terminals in the hippocampal mossy fibers and the number of NeuN-labeled neurons in hippocampal CA3 areas marked by rectangles (Zoomed-in images shown in the lower row of each panel in A ) were quantified and normalized to those of AAV-mCherry-injected non-Tg control mice. Data were collected from three mice per group. ( C ) Contextual fear conditioning task performed in 7-month-old non-Tg and tauP301S Tg male mice infected with AAV-mCherry or AAV-mCherry-hTBC1D15 ( n = 15-17 male mice per group). Data were expressed as the mean ± SEM and analyzed using linear mixed-effects models ( B) or by one-way ANOVA, followed by Sidak’s post-hoc test ( C ). Scale bars ( A) : 20 μm (upper row, SYP), 250 μm (upper row, NeuN), and 25 μm (lower row, NeuN).

Article Snippet: The AAV2/9-mCherry, AAV2/9-mCherry-hTBC1D15, and AAV2/9-mCherry-hTBC1D15 D397A mutant viruses were produced by Vector BioLabs.

Techniques: Labeling, Injection, Control, Infection

Journal: eLife

Article Title: Control of Slc7a5 sensitivity by the voltage-sensing domain of Kv1 channels

doi: 10.7554/eLife.54916

Figure Lengend Snippet:

Article Snippet: Gene , mCherry , Addgene , 30125 , .

Techniques: Transfection, Recombinant, Sequencing, Plasmid Preparation

Journal: iScience

Article Title: Horizontal and vertical gene transfer shape the plasmid host range in surface-associated microbial systems

doi: 10.1016/j.isci.2026.115299

Figure Lengend Snippet: Hypothesis and experiment system (A) We hypothesize that vertical and horizontal gene transfer (VGT and HGT) are influenced by the characteristics of the potential recipient cell types and determine the proliferation and diversity of transconjugant cells. Because the potential recipient community comprises multiple cell types with varying growth traits and conjugation probabilities, we expect the resulting composition of transconjugant cells to be shaped by these cell type-specific traits. (B) Our experimental system consists of E . coli MG1655 lacI q -pLpp-mCherry as the plasmid donor strain and pB10 as the focal plasmid. pB10 donor cells express RFP from the chromosome and transconjugants express GFP from pB10.

Article Snippet: MBP- mCherry expression plasmid (Amp R ) , Addgene , Plasmid# 29747.

Techniques: Conjugation Assay, Plasmid Preparation

Journal: iScience

Article Title: Horizontal and vertical gene transfer shape the plasmid host range in surface-associated microbial systems

doi: 10.1016/j.isci.2026.115299

Figure Lengend Snippet: Transconjugant proportions and diversities after surface-associated conjugation assays for different environmental conditions (A) Proportion of transconjugant cells relative to total cells after surface-associated conjugation assays using the WWTP community as the potential recipient cell population. We conducted conjugation assays on 1×SWW, 10×SWW, or LB agar plates using E . coli MG1655 lacI q -pLpp-mCherry as the pB10 donor strain. (B) Relative abundances of bacterial class in the total potential recipient cell population (T) and the transconjugant cell population (TC) as identified by 16S rRNA gene sequencing. We separated and identified TC cells using FC-FACS-sorting of GFP-positive cells. (C) Normalized Shannon index of the transconjugant populations after surface-associated conjugation assays on 1×SWW, 10×SWW, or LB agar plates. We normalized the Shannon index of the TC populations to their corresponding T populations. (D) Principal coordinate analysis (PCoA) based on weighted UniFrac distances of T and TC populations after surface-associated conjugation assays on 1×SWW, 10×SWW, or LB agar plates. (E) Phylogenetic tree of transconjugant ASVs detected after surface-associated conjugation assays on 1×SWW, 10×SWW, or LB agar plates. The outer colored box denotes the bacterial phylum of each ASV, corresponding to the phylum-level groupings shown in panel (B). The inner heatmap box aligned with each tip shows the log 10 fold-changes in ASV abundance (TC relative to T) across the three conditions. For (A and C), each point is an independent biological replicate ( n = 3), horizontal bars are the means, error bars are ±1 standard deviation, and asterisks indicate statistically significant differences between the means based on two-way ANOVA with Holm correction (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns = not significant). For (D), each point is an independent biological replicate ( n = 3).

Article Snippet: MBP- mCherry expression plasmid (Amp R ) , Addgene , Plasmid# 29747.

Techniques: Conjugation Assay, Sequencing, Standard Deviation

Journal: iScience

Article Title: Horizontal and vertical gene transfer shape the plasmid host range in surface-associated microbial systems

doi: 10.1016/j.isci.2026.115299

Figure Lengend Snippet: Transconjugant growth during surface-associated conjugation assays for different environmental conditions (A) Representative fluorescence microscopy images of transconjugant cells during surface-associated conjugation assays on LB agar plates. E . coli MG1655 lacI q -pLpp-mCherry is the pB10 donor strain and show red fluorescence. Transconjugant cells are green. The time indicated in the images refers to the point at which transconjugant cells first became detectable. (B) Normalized microcolony area ( A / a 0 ) plotted as a function of time during the surface-associated conjugation assays on LB agar plates. A is the total microcolony area and a 0 is the initial transconjugant area. Connected data points are for individual colonies ( n = 12). (C) Microcolony area at the endpoint of the mating assay (t = 24 h) for different environmental conditions. The half-violin and scatterplots present the sample distribution and individual microcolony measurements for surface-associated conjugation assays on different medium (n 1xSWW = 880, n 10xSWW = 664, n LB = 1,070, for microcolony number). We performed each experiment at least three independent experiments. Horizontal bars are the mean microcolony areas, error bars are the 99% confidence intervals, and asterisks indicate statistically significant differences between the means based on two-way ANOVA with Holm correction (∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, ns = not significant).

Article Snippet: MBP- mCherry expression plasmid (Amp R ) , Addgene , Plasmid# 29747.

Techniques: Conjugation Assay, Fluorescence, Microscopy