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anti-pla2g6 antibody 376563 (Santa Cruz Biotechnology)

Name: anti-pla2g6 antibody 376563
Brand: Santa Cruz Biotechnology
Rating: 90 (1 reviews)

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Santa Cruz Biotechnology anti-pla2g6 antibody 376563
Anti Pla2g6 Antibody 376563, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews

anti-pla2g6 antibody 376563 - by Bioz Stars, 2026-03

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Journal: iScience

Article Title: Phospholipase PLA2G7 is complementary to GPX4 in mitigating punicic-acid-induced ferroptosis in prostate cancer cells

doi: 10.1016/j.isci.2024.109774

Figure Lengend Snippet:

Article Snippet: Mouse polyclonal anti-PLA2G6 , Santa Cruz antibody , Cat# sc376563 RRID: AB_ 11150308.

Techniques: Virus, Control, Expressing, Plasmid Preparation, Recombinant, Western Blot, Transfection, Saline, Protease Inhibitor, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Software, RNA Sequencing

Figure 1. Ceramide accumulation, lysosomal expansion, and mitochondrial defects in INAD patient-derived NPCs and DA neurons. (A) PLA2G6 is expressed in iPSCs and NPCs using the sc-376563 antibody. The control iPSCs and NPCs were generated by reprograming a fibroblast line from a healthy person (GM23815; Coriell Institute). The 29-3 iPSCs and NPCs were generated from lymphoblasts from an INAD patient in Family 2 (Figure 1—figure supplement 1D). Actin was used as a loading control. (B) GlcCer levels in DA neurons (images in Figure 1—figure supplement

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/elife.82555

Figure Lengend Snippet: Figure 1. Ceramide accumulation, lysosomal expansion, and mitochondrial defects in INAD patient-derived NPCs and DA neurons. (A) PLA2G6 is expressed in iPSCs and NPCs using the sc-376563 antibody. The control iPSCs and NPCs were generated by reprograming a fibroblast line from a healthy person (GM23815; Coriell Institute). The 29-3 iPSCs and NPCs were generated from lymphoblasts from an INAD patient in Family 2 (Figure 1—figure supplement 1D). Actin was used as a loading control. (B) GlcCer levels in DA neurons (images in Figure 1—figure supplement

Article Snippet: We tested the specificity of three commercially available PLA2G6 antibodies and found one antibody that recognizes PAL2G6 (sc- 376563 Santa Cruz Biotechnology; Figure 1—figure supplement 1A- B).

Techniques: Derivative Assay, Control, Generated

Figure 3. Ambroxol, Azoramide, Desipramine, and Genistein alleviate neurodegenerative phenotypes in INAD flies and patient-derived NPCs. (A) Bang-sensitivity was used as a primary readout to select drugs that suppress neurodegeneration. Bang-sensitivity of control or INAD flies fed with the indicated drugs. Error bars represent SEM (n=3; 20 flies per assay). Redline highlights the time required for INAD flies to recover from bang-induced paralysis. Red ‘*‘ indicates drugs that significantly suppress bang-sensitivity. Black ‘*‘ indicates drugs that significantly promote bang-sensitivity (the p values were calculated between iPLA2-VIA∆174 and the indicated treatment). (B) Using INAD patient-derived NPCs to select drugs that suppress LAMP2 accumulation. PLA2G6 antibody was used to detect the endogenous PLA2G6 in the indicated cellular lysates. The intensity of the LAMP2/Actin is quantified at (C) (n=3). (D) Drugs that suppress LAMP2 accumulation also restore ATP levels. The relative amounts of ATP are measured in the indicated

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/elife.82555

Figure Lengend Snippet: Figure 3. Ambroxol, Azoramide, Desipramine, and Genistein alleviate neurodegenerative phenotypes in INAD flies and patient-derived NPCs. (A) Bang-sensitivity was used as a primary readout to select drugs that suppress neurodegeneration. Bang-sensitivity of control or INAD flies fed with the indicated drugs. Error bars represent SEM (n=3; 20 flies per assay). Redline highlights the time required for INAD flies to recover from bang-induced paralysis. Red ‘*‘ indicates drugs that significantly suppress bang-sensitivity. Black ‘*‘ indicates drugs that significantly promote bang-sensitivity (the p values were calculated between iPLA2-VIA∆174 and the indicated treatment). (B) Using INAD patient-derived NPCs to select drugs that suppress LAMP2 accumulation. PLA2G6 antibody was used to detect the endogenous PLA2G6 in the indicated cellular lysates. The intensity of the LAMP2/Actin is quantified at (C) (n=3). (D) Drugs that suppress LAMP2 accumulation also restore ATP levels. The relative amounts of ATP are measured in the indicated

Techniques: Derivative Assay, Control

Figure 4. Expression of human PLA2G6 restores lysosomal and mitochondrial morphology defects in INAD patient-derived NPC lines. (A) Vectors/ constructs. (B) Expression levels of the constructs (A) in 293T cells. PLA2G6 antibody was used to detect the endogenous PLA2G6 of cellular lysates. * represents endogenous PLA2G6. # indicates a nonspecific band. All AAV constructs were used to infect 293T cell using a MOI of 1000. The MOI of the Lenti-viral-based construct was not determined. (C) Expression levels of the illustrated constructs (A) in NPCs. AAV constructs were used to

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/elife.82555

Figure Lengend Snippet: Figure 4. Expression of human PLA2G6 restores lysosomal and mitochondrial morphology defects in INAD patient-derived NPC lines. (A) Vectors/ constructs. (B) Expression levels of the constructs (A) in 293T cells. PLA2G6 antibody was used to detect the endogenous PLA2G6 of cellular lysates. * represents endogenous PLA2G6. # indicates a nonspecific band. All AAV constructs were used to infect 293T cell using a MOI of 1000. The MOI of the Lenti-viral-based construct was not determined. (C) Expression levels of the illustrated constructs (A) in NPCs. AAV constructs were used to

Techniques: Expressing, Derivative Assay, Construct

Figure 7. ICV and IV injection of AAV-PHP.eB-EF1a-PLA2G6 reduces GlcCer and LAMP2 accumulation and restores the number of Vps35 punctae. (A) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. GlcCer antibody (green; arrows) was used to assess the levels of GlcCer. Scale bar=100 µm. All assays were conducted blind for genotypes and treatments. (B) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. LAMP2 antibody (green; arrows) labels lysosomes in the Purkinje cells. Scale bar=20 µm. (C) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. Vps35 antibody (green; arrows). Scale bar=20 µm. Representative images are shown in this figure. DAPI (blue) labels nuclei. Representative images are shown in this figure. Quantifications are shown next to the images (n=3 for wild-type and Pla2g6KO/G373R mice; n=2 for Pla2g6KO/G373R-injected mice). Error bars represent SEM; *p<0.05; **p<0.01. ICV, intracerebroventricular; IV, intravenous.

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/elife.82555

Figure Lengend Snippet: Figure 7. ICV and IV injection of AAV-PHP.eB-EF1a-PLA2G6 reduces GlcCer and LAMP2 accumulation and restores the number of Vps35 punctae. (A) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. GlcCer antibody (green; arrows) was used to assess the levels of GlcCer. Scale bar=100 µm. All assays were conducted blind for genotypes and treatments. (B) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. LAMP2 antibody (green; arrows) labels lysosomes in the Purkinje cells. Scale bar=20 µm. (C) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. Vps35 antibody (green; arrows). Scale bar=20 µm. Representative images are shown in this figure. DAPI (blue) labels nuclei. Representative images are shown in this figure. Quantifications are shown next to the images (n=3 for wild-type and Pla2g6KO/G373R mice; n=2 for Pla2g6KO/G373R-injected mice). Error bars represent SEM; *p<0.05; **p<0.01. ICV, intracerebroventricular; IV, intravenous.

Techniques: IV Injection, Staining, Injection

The injected Pla2g6 KO/G373R mice (at ~P300) behaved relatively normal in their home cage.

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: The injected Pla2g6 KO/G373R mice (at ~P300) behaved relatively normal in their home cage.

Article Snippet: Primary antibodies used in this study were as follows: mouse anti-PLA2G6 antibody (Santa Cruz Biotechnology; sc376563), rabbit anti-PLA2G6 antibody (Sigma-Aldrich; SAB4200129), rabbit anti-PLA2G6 antibody (Sigma-Aldrich; HPA001171), mouse anti-Actin (ICN691001, Thermo Fisher Scientific), and rabbit anti-LAMP2 antibody (Abcam; ab18528).

Techniques:

( A ) Identification of commercially available PLA2G6 antibodies that specifically recognize endogenous levels of PLA2G6 in HEK-293T cellular lysate. The SAB200129 antibody recognizes a major band at the predicted molecular weight (85/88 kDa) of PLA2G6. The sc-376563 antibody detects five major bands with one band at ~85/88 kDa. HPA001171 detects many bands at similar intensity, hence is not a proper antibody to be used. ( B, C ) sc-376563 specifically recognizes endogenous PLA2G6 in HEK-293T cells ( B ) and a control skin fibroblast line ( C ). The 85/88 kDa (the predicted molecular weight of PLA2G6) band detected by sc-376563 is significantly reduced upon PLA2G6 shRNA treatments, whereas the single band detected by SAB200129 does not. ( D ) PLA2G6 is not expressed in most skin fibroblasts shown in . The experiments were conducted in blind of genotype. sc-376563 antibody was used to label PLA2G6. LAMP2 antibody was used to assess lysosomal accumulation. Actin was used as a loading control. (E) INAD patient skin fibroblasts exhibit highly variable mitochondrial phenotypes. Representative images are shown in this figure. Scale bar=1 µm. Figure 1—figure supplement 1—source data 1. INAD patient skin fibroblast lines used in this study. Figure 1—figure supplement 1—source data 2. Raw gel images for . Figure 1—figure supplement 1—source data 3. Skin fibroblasts derived from INAD patients exhibit highly variable phenotypes.

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: ( A ) Identification of commercially available PLA2G6 antibodies that specifically recognize endogenous levels of PLA2G6 in HEK-293T cellular lysate. The SAB200129 antibody recognizes a major band at the predicted molecular weight (85/88 kDa) of PLA2G6. The sc-376563 antibody detects five major bands with one band at ~85/88 kDa. HPA001171 detects many bands at similar intensity, hence is not a proper antibody to be used. ( B, C ) sc-376563 specifically recognizes endogenous PLA2G6 in HEK-293T cells ( B ) and a control skin fibroblast line ( C ). The 85/88 kDa (the predicted molecular weight of PLA2G6) band detected by sc-376563 is significantly reduced upon PLA2G6 shRNA treatments, whereas the single band detected by SAB200129 does not. ( D ) PLA2G6 is not expressed in most skin fibroblasts shown in . The experiments were conducted in blind of genotype. sc-376563 antibody was used to label PLA2G6. LAMP2 antibody was used to assess lysosomal accumulation. Actin was used as a loading control. (E) INAD patient skin fibroblasts exhibit highly variable mitochondrial phenotypes. Representative images are shown in this figure. Scale bar=1 µm. Figure 1—figure supplement 1—source data 1. INAD patient skin fibroblast lines used in this study. Figure 1—figure supplement 1—source data 2. Raw gel images for . Figure 1—figure supplement 1—source data 3. Skin fibroblasts derived from INAD patients exhibit highly variable phenotypes.

Techniques: Molecular Weight, Control, shRNA, Derivative Assay

( A ) PLA2G6 is expressed in iPSCs and NPCs using the sc-376563 antibody. The control iPSCs and NPCs were generated by reprograming a fibroblast line from a healthy person (GM23815; Coriell Institute). The 29-3 iPSCs and NPCs were generated from lymphoblasts from an INAD patient in Family 2 . Actin was used as a loading control. ( B ) GlcCer levels in DA neurons (images in ) (n=8). ( C ) Lysosomal expansion in DA neurons (images in ) (n=8–9). ( D ) Cells with mitochondrial defects (images in ) (n=6–8). ( E ) The lysosomal and mitochondrial defects are rescued in edited INAD patient-derived NPCs. Immunofluorescence staining of the indicated INAD patient-derived NPCs. LAMP2 antibody (green; arrows) and TOMM20 antibody (red) were used to label lysosomes and mitochondria, respectively. DAPI (blue) labels cell nuclei. Scale bar=5 µm. The quantifications of the average lysosome number per cell (n=10) and the relative mitochondrial length per cell (n=10) are presented next to the images. ( F ) Lysosomal LAMP2 accumulation is reduced in edited INAD patient-derived NPCs. PLA2G6 antibody was used to detect the endogenous PLA2G6 in the indicated cellular lysates. LAMP2 antibody was used to assess lysosomal accumulation. Actin was used as a loading control. The intensity of the LAMP2/Actin is quantified at the right (n=3). ( G ) ATP levels are restored in edited INAD patient-derived NPCs. The relative amount of ATP are quantified in the indicated NPC lines (n=3). ( H ) Ceramide levels are reduced in the edited INAD patient-derived NPCs. The levels of the indicated ceramide and its derivatives are measured by lipidomic assays (n=3). Representative images are shown in this figure. Error bars represent SEM; *p<0.05; **p<0.01; ***p<0.001. INAD, infantile neuroaxonal dystrophy; NPC, neural progenitor cell. Figure 1—source data 1. PLA2G6 is expressed in iPSCs and NPCs. Figure 1—source data 2. The lysosomal defects are rescued in edited INAD patient-derived NPCs. Figure 1—source data 3. Lipidomic assay of INAD patient derived NPCs. Figure 1—source data 4. Raw gel images for .

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: ( A ) PLA2G6 is expressed in iPSCs and NPCs using the sc-376563 antibody. The control iPSCs and NPCs were generated by reprograming a fibroblast line from a healthy person (GM23815; Coriell Institute). The 29-3 iPSCs and NPCs were generated from lymphoblasts from an INAD patient in Family 2 . Actin was used as a loading control. ( B ) GlcCer levels in DA neurons (images in ) (n=8). ( C ) Lysosomal expansion in DA neurons (images in ) (n=8–9). ( D ) Cells with mitochondrial defects (images in ) (n=6–8). ( E ) The lysosomal and mitochondrial defects are rescued in edited INAD patient-derived NPCs. Immunofluorescence staining of the indicated INAD patient-derived NPCs. LAMP2 antibody (green; arrows) and TOMM20 antibody (red) were used to label lysosomes and mitochondria, respectively. DAPI (blue) labels cell nuclei. Scale bar=5 µm. The quantifications of the average lysosome number per cell (n=10) and the relative mitochondrial length per cell (n=10) are presented next to the images. ( F ) Lysosomal LAMP2 accumulation is reduced in edited INAD patient-derived NPCs. PLA2G6 antibody was used to detect the endogenous PLA2G6 in the indicated cellular lysates. LAMP2 antibody was used to assess lysosomal accumulation. Actin was used as a loading control. The intensity of the LAMP2/Actin is quantified at the right (n=3). ( G ) ATP levels are restored in edited INAD patient-derived NPCs. The relative amount of ATP are quantified in the indicated NPC lines (n=3). ( H ) Ceramide levels are reduced in the edited INAD patient-derived NPCs. The levels of the indicated ceramide and its derivatives are measured by lipidomic assays (n=3). Representative images are shown in this figure. Error bars represent SEM; *p<0.05; **p<0.01; ***p<0.001. INAD, infantile neuroaxonal dystrophy; NPC, neural progenitor cell. Figure 1—source data 1. PLA2G6 is expressed in iPSCs and NPCs. Figure 1—source data 2. The lysosomal defects are rescued in edited INAD patient-derived NPCs. Figure 1—source data 3. Lipidomic assay of INAD patient derived NPCs. Figure 1—source data 4. Raw gel images for .

Techniques: Control, Generated, Derivative Assay, Immunofluorescence, Staining

( A ) Generation of INAD patient-derived NPCs and DA neurons. ( B–D ) Patient and isogenic control lines differentiate into comparable ventral midbrain floor plate neuron progenitor cells. ( B, C ) Day 22 cultures from an INAD patient-derived PLA2G6 mutant iPSC clone 29-1, isogenic PLA2G6 corrected iPSC clone 29-2 (derived from 29-1), and INAD patient-derived PLA2G6 mutant iPSC clone 29-3. ( B ) Uniformly express pan NPC intermediate filament marker NESTIN (green), nuclear (DAPI, blue), floor plate markers FOXA2 (red), and vM FP marker LMX1A. Scale bar = 200 µm ( C ) Sister wells show nuclei (DAPI, blue), uniformly express rostral-to-midbrain marker OTX2 (red) and do not express forebrain marker FOXG1 (yellow). 3×3 montage of representative randomly sampled 10× Phenix confocal fields. ( D ) The differentiated DA neurons are TH (red), NESTIN (green), and MAP2 (purple) positive. Representative images are shown in this figure. Scale bars = 20 µm. DA, dopaminergic; INAD, infantile neuroaxonal dystrophy; NPC, neural progenitor cell.

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: ( A ) Generation of INAD patient-derived NPCs and DA neurons. ( B–D ) Patient and isogenic control lines differentiate into comparable ventral midbrain floor plate neuron progenitor cells. ( B, C ) Day 22 cultures from an INAD patient-derived PLA2G6 mutant iPSC clone 29-1, isogenic PLA2G6 corrected iPSC clone 29-2 (derived from 29-1), and INAD patient-derived PLA2G6 mutant iPSC clone 29-3. ( B ) Uniformly express pan NPC intermediate filament marker NESTIN (green), nuclear (DAPI, blue), floor plate markers FOXA2 (red), and vM FP marker LMX1A. Scale bar = 200 µm ( C ) Sister wells show nuclei (DAPI, blue), uniformly express rostral-to-midbrain marker OTX2 (red) and do not express forebrain marker FOXG1 (yellow). 3×3 montage of representative randomly sampled 10× Phenix confocal fields. ( D ) The differentiated DA neurons are TH (red), NESTIN (green), and MAP2 (purple) positive. Representative images are shown in this figure. Scale bars = 20 µm. DA, dopaminergic; INAD, infantile neuroaxonal dystrophy; NPC, neural progenitor cell.

Techniques: Derivative Assay, Control, Mutagenesis, Marker

( A ) Both Pla2g6 G373R/G373R and Pla2g6 KO/G373R mice show severe rotarod defect. Rotarod performance of mice with the indicated genotypes was measured weekly. Wild-type (n=6); Pla2g6 +/G373R (n=10); Pla2g6 G373R/G373R (n=7); Pla2g6 KO/+ (n=4); and Pla2g6 KO/G373R (n=7). Error bars represent SEM. ( B ) Pla2g6 G373R/G373R and Pla2g6 KO/G373R mice fantile neuroaxonal dystrophy. A disease characterized by altered terminal axons and synaptic enshow a severe reduction of lifespan. Wild-type (n=6); Pla2g6 G373R/G373R (n=9); and Pla2g6 KO/G373R (n=8). ( C ) Accumulation of GlcCer in PLA2G6 KO/G373R and PLA2G6 G373R/G373R mice. Immunofluorescent staining of mouse cerebella and midbrain regions of the indicated genotypes. GlcCer antibody (green) was used to assess the levels of GlcCer. TH (Tyrosine Hydroxylase, red) antibody labels DA neurons in the midbrain region. Scale bar=100 µm. All assays were conducted in blind of genotypes and treatments. ( D ) Lysosomal expansion in INAD in Pla2g6 KO/G373R and Pla2g6 G373R/G373R mice. Immunofluorescent staining of mouse cerebella and midbrain regions of the indicated genotypes. LAMP1 antibody (green; arrows) labels lysosomes. DAPI (blue) labels nuclei. Representative images are shown in this figure. Quantifications are next to the images (n=3). Scale bar=10 µm (cerebellum) or 5 µm (midbrain). DA, dopaminergic; INAD, infantile neuroaxonal dystrophy. Figure 2—source data 1. Lipidomic assay of the cerebellum of INAD mice.

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: ( A ) Both Pla2g6 G373R/G373R and Pla2g6 KO/G373R mice show severe rotarod defect. Rotarod performance of mice with the indicated genotypes was measured weekly. Wild-type (n=6); Pla2g6 +/G373R (n=10); Pla2g6 G373R/G373R (n=7); Pla2g6 KO/+ (n=4); and Pla2g6 KO/G373R (n=7). Error bars represent SEM. ( B ) Pla2g6 G373R/G373R and Pla2g6 KO/G373R mice fantile neuroaxonal dystrophy. A disease characterized by altered terminal axons and synaptic enshow a severe reduction of lifespan. Wild-type (n=6); Pla2g6 G373R/G373R (n=9); and Pla2g6 KO/G373R (n=8). ( C ) Accumulation of GlcCer in PLA2G6 KO/G373R and PLA2G6 G373R/G373R mice. Immunofluorescent staining of mouse cerebella and midbrain regions of the indicated genotypes. GlcCer antibody (green) was used to assess the levels of GlcCer. TH (Tyrosine Hydroxylase, red) antibody labels DA neurons in the midbrain region. Scale bar=100 µm. All assays were conducted in blind of genotypes and treatments. ( D ) Lysosomal expansion in INAD in Pla2g6 KO/G373R and Pla2g6 G373R/G373R mice. Immunofluorescent staining of mouse cerebella and midbrain regions of the indicated genotypes. LAMP1 antibody (green; arrows) labels lysosomes. DAPI (blue) labels nuclei. Representative images are shown in this figure. Quantifications are next to the images (n=3). Scale bar=10 µm (cerebellum) or 5 µm (midbrain). DA, dopaminergic; INAD, infantile neuroaxonal dystrophy. Figure 2—source data 1. Lipidomic assay of the cerebellum of INAD mice.

Techniques: Staining

( A ) Abnormal mitochondrial morphology in Purkinje cells of Pla2g6 G373R/G373R mice. The yellow dotted line circles a representative Purkinje cell. Scale bar=2 µm. The boxed regions are enlarged at right. Scale bar=800 nm. Mt, mitochondria; N, nuclei; TVS, tubulovesicular structure. ( B ) Pla2g6 G373R/G373R mice show disrupted mitochondria and increased MVB and TVS. ( C ) The quantification of the disrupted mitochondria, number of MVB or TVS in the Purkinje cells of Pla2g6 G373R/G373R mice. ( D ) The levels of the indicated ceramides in the cerebellum of KO/G373R mice (n=3). Representative images are shown in this figure. Error bars represent SEM. ***p<0.001; NS, not significant.

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: ( A ) Abnormal mitochondrial morphology in Purkinje cells of Pla2g6 G373R/G373R mice. The yellow dotted line circles a representative Purkinje cell. Scale bar=2 µm. The boxed regions are enlarged at right. Scale bar=800 nm. Mt, mitochondria; N, nuclei; TVS, tubulovesicular structure. ( B ) Pla2g6 G373R/G373R mice show disrupted mitochondria and increased MVB and TVS. ( C ) The quantification of the disrupted mitochondria, number of MVB or TVS in the Purkinje cells of Pla2g6 G373R/G373R mice. ( D ) The levels of the indicated ceramides in the cerebellum of KO/G373R mice (n=3). Representative images are shown in this figure. Error bars represent SEM. ***p<0.001; NS, not significant.

Techniques:

( A ) Bang-sensitivity was used as a primary readout to select drugs that suppress neurodegeneration. Bang-sensitivity of control or INAD flies fed with the indicated drugs. Error bars represent SEM (n=3; 20 flies per assay). Redline highlights the time required for INAD flies to recover from bang-induced paralysis. Red ‘*‘ indicates drugs that significantly suppress bang-sensitivity. Black ‘*‘ indicates drugs that significantly promote bang-sensitivity (the p values were calculated between iPLA2-VIA ∆174 and the indicated treatment). (B) Using INAD patient-derived NPCs to select drugs that suppress LAMP2 accumulation. PLA2G6 antibody was used to detect the endogenous PLA2G6 in the indicated cellular lysates. The intensity of the LAMP2/Actin is quantified at (C) (n=3). (D) Drugs that suppress LAMP2 accumulation also restore ATP levels. The relative amounts of ATP are measured in the indicated NPC lines with or without the treatment of the selected drugs (n=3). Error bars represent SEM; *p<0.05; **p<0.01; ***p<0.001; NS, not significant (the p values were calculated between the untreated patient NPCs (29-1) and the indicated treatment). References in : ( ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ). INAD, infantile neuroaxonal dystrophy; NPC, neural progenitor cell. Figure 3—source data 1. Ambroxol, Azoramide, Desipramine, or Genistein reduce LAMP2 levels in patient derived NPCs. Figure 3—source data 2. Selected drugs tested in an INAD fly model. Figure 3—source data 3. Raw gel images for .

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: ( A ) Bang-sensitivity was used as a primary readout to select drugs that suppress neurodegeneration. Bang-sensitivity of control or INAD flies fed with the indicated drugs. Error bars represent SEM (n=3; 20 flies per assay). Redline highlights the time required for INAD flies to recover from bang-induced paralysis. Red ‘*‘ indicates drugs that significantly suppress bang-sensitivity. Black ‘*‘ indicates drugs that significantly promote bang-sensitivity (the p values were calculated between iPLA2-VIA ∆174 and the indicated treatment). (B) Using INAD patient-derived NPCs to select drugs that suppress LAMP2 accumulation. PLA2G6 antibody was used to detect the endogenous PLA2G6 in the indicated cellular lysates. The intensity of the LAMP2/Actin is quantified at (C) (n=3). (D) Drugs that suppress LAMP2 accumulation also restore ATP levels. The relative amounts of ATP are measured in the indicated NPC lines with or without the treatment of the selected drugs (n=3). Error bars represent SEM; *p<0.05; **p<0.01; ***p<0.001; NS, not significant (the p values were calculated between the untreated patient NPCs (29-1) and the indicated treatment). References in : ( ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ). INAD, infantile neuroaxonal dystrophy; NPC, neural progenitor cell. Figure 3—source data 1. Ambroxol, Azoramide, Desipramine, or Genistein reduce LAMP2 levels in patient derived NPCs. Figure 3—source data 2. Selected drugs tested in an INAD fly model. Figure 3—source data 3. Raw gel images for .

Techniques: Control, Derivative Assay

( A ) The ERG trace of the indicated genotypes and treatments. iPLA2-VIA ∆174 : the INAD flies; GR; iPLA2-VIA ∆174 : genomic rescued INAD flies (control) . ( B ) Quantifications of the LCRP amplitudes. ( C ) Quantifications of the on-transient amplitudes. ( D ) Ambroxol, Azoramide, Desipramine or Genistein reduce the loss of photoreceptor phenotype in INAD flies. Toluidine blue staining of the ommatidia of the flies with the indicated genotypes and treatment. Arrows indicate vacuoles. The number of photoreceptor per ommatidia is quantified in ( E ) (n=10). Scale bar=10 µm. Representative images are shown in this figure. Error bars represent SEM; ***p<0.001 (the p values were calculated between the untreated (No Drug) and the indicated column). ERG, electroretinogram; INAD, infantile neuroaxonal dystrophy; LCRP, light coincident receptor potential.

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: ( A ) The ERG trace of the indicated genotypes and treatments. iPLA2-VIA ∆174 : the INAD flies; GR; iPLA2-VIA ∆174 : genomic rescued INAD flies (control) . ( B ) Quantifications of the LCRP amplitudes. ( C ) Quantifications of the on-transient amplitudes. ( D ) Ambroxol, Azoramide, Desipramine or Genistein reduce the loss of photoreceptor phenotype in INAD flies. Toluidine blue staining of the ommatidia of the flies with the indicated genotypes and treatment. Arrows indicate vacuoles. The number of photoreceptor per ommatidia is quantified in ( E ) (n=10). Scale bar=10 µm. Representative images are shown in this figure. Error bars represent SEM; ***p<0.001 (the p values were calculated between the untreated (No Drug) and the indicated column). ERG, electroretinogram; INAD, infantile neuroaxonal dystrophy; LCRP, light coincident receptor potential.

Techniques: Control, Staining

( A ) Vectors/constructs. ( B ) Expression levels of the constructs ( A ) in 293T cells. PLA2G6 antibody was used to detect the endogenous PLA2G6 of cellular lysates. * represents endogenous PLA2G6. # indicates a nonspecific band. All AAV constructs were used to infect 293T cell using a MOI of 1000. The MOI of the Lenti-viral-based construct was not determined. ( C ) Expression levels of the illustrated constructs ( A ) in NPCs. AAV constructs were used to infect NPCs at a MOI of 1000. The MOI of the Lenti-viral-based construct was not determined. The intensity of LAMP2/Actin is quantified below (n=3). ( D ) Expression of human PLA2G6 restores mitochondrial morphology defects in INAD patient-derived NPCs. Arrows indicate the fragmented and enlarged mitochondria. Arrowheads indicate the normal elongated network of mitochondria. ( E ) Expression of human PLA2G6 restores mitochondrial morphology and ATP levels. The length of the mitochondria in ( D ) is quantified in ( E ) (n=10; upper panel). The ATP levels are measure in the lower panel (n=3). Representative images are shown in this figure. Error bars represent SEM; *p<0.05; ***p<0.001 (the p values were calculated between the untreated patient NPCs (29-1) and the indicated treatment). INAD, infantile neuroaxonal dystrophy; MOI, multiplicity of infection; NPC, neural progenitor cell. Figure 4—source data 1. Expression of human PLA2G6 in INAD patient derived NPCs. Figure 4—source data 2. Expression of human PLA2G6 restores lysosomal defects in INAD patient-derived NPCs. Figure 4—source data 3. Raw gel images for .

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: ( A ) Vectors/constructs. ( B ) Expression levels of the constructs ( A ) in 293T cells. PLA2G6 antibody was used to detect the endogenous PLA2G6 of cellular lysates. * represents endogenous PLA2G6. # indicates a nonspecific band. All AAV constructs were used to infect 293T cell using a MOI of 1000. The MOI of the Lenti-viral-based construct was not determined. ( C ) Expression levels of the illustrated constructs ( A ) in NPCs. AAV constructs were used to infect NPCs at a MOI of 1000. The MOI of the Lenti-viral-based construct was not determined. The intensity of LAMP2/Actin is quantified below (n=3). ( D ) Expression of human PLA2G6 restores mitochondrial morphology defects in INAD patient-derived NPCs. Arrows indicate the fragmented and enlarged mitochondria. Arrowheads indicate the normal elongated network of mitochondria. ( E ) Expression of human PLA2G6 restores mitochondrial morphology and ATP levels. The length of the mitochondria in ( D ) is quantified in ( E ) (n=10; upper panel). The ATP levels are measure in the lower panel (n=3). Representative images are shown in this figure. Error bars represent SEM; *p<0.05; ***p<0.001 (the p values were calculated between the untreated patient NPCs (29-1) and the indicated treatment). INAD, infantile neuroaxonal dystrophy; MOI, multiplicity of infection; NPC, neural progenitor cell. Figure 4—source data 1. Expression of human PLA2G6 in INAD patient derived NPCs. Figure 4—source data 2. Expression of human PLA2G6 restores lysosomal defects in INAD patient-derived NPCs. Figure 4—source data 3. Raw gel images for .

Techniques: Construct, Expressing, Derivative Assay, Infection

A ( a, b ) The number of Vps35 punctae is higher in the genetically corrected NPCs (29-2). Immunofluorescent staining of NPCs with the indicated genotypes and treatments. Vps35 antibody (green; arrows) labels retromers. DAPI (blue) labels nuclei. ( c–f ) The number of Vps35 punctae is not affected in the patient-derived NPCs (29-1) treated with the selected drugs. ( g–i ) The expression of human PLA2G6 restores the number of Vps35 punctae in INAD patient-derived NPCs. ( B ) Quantifications of the Vps35 punctae in ( A ) (n=8). Representative images are shown in this figure. Scale bar=20 µm. Error bars represent SEM; ***p<0.001; NS, not significant (the p values were calculated between the untreated patient NPCs (29-1) and the indicated treatment). INAD, infantile neuroaxonal dystrophy; NPC, neural progenitor cell.

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: A ( a, b ) The number of Vps35 punctae is higher in the genetically corrected NPCs (29-2). Immunofluorescent staining of NPCs with the indicated genotypes and treatments. Vps35 antibody (green; arrows) labels retromers. DAPI (blue) labels nuclei. ( c–f ) The number of Vps35 punctae is not affected in the patient-derived NPCs (29-1) treated with the selected drugs. ( g–i ) The expression of human PLA2G6 restores the number of Vps35 punctae in INAD patient-derived NPCs. ( B ) Quantifications of the Vps35 punctae in ( A ) (n=8). Representative images are shown in this figure. Scale bar=20 µm. Error bars represent SEM; ***p<0.001; NS, not significant (the p values were calculated between the untreated patient NPCs (29-1) and the indicated treatment). INAD, infantile neuroaxonal dystrophy; NPC, neural progenitor cell.

Techniques: Staining, Derivative Assay, Expressing

( A ) Rotarod defect in Pla2g6 KO/G373R mice (genotype: Pla2g6 KO/G373R ) (n=6) are reduced by pre-symptomatic ( P40 ) ICV + IV injection (n=5), but not ICV only (n=2). Wild-type (n=5); Pla2g6 KO/G373R (n=10). Rotarod performance of mice with the indicated genotypes was measured weekly. ( B ) Pre-symptomatic ( P40 ) ICV + IV injection of AAV-PHP.eB-hPLA2G6 stabilizes body weight of Pla2g6 KO/G373R mice. Wild-type (n=5); Pla2g6 KO/G373R (n=6); Pla2g6 KO/G373R injected (n=3). ( C ) Pre-symptomatic ( P40 ) ICV + IV injection of AAV-PHP.eB-PLA2G6 prolongs lifespan of the Pla2g6 KO/G373R mice. Pla2g6 KO/G373R (n=8); Pla2g6 KO/G373R injected (n=8). Error bars represent SEM. * P<0.05; ** P<0.01; *** P<0.001. All assays were conducted in blind of genotypes and treatments.

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: ( A ) Rotarod defect in Pla2g6 KO/G373R mice (genotype: Pla2g6 KO/G373R ) (n=6) are reduced by pre-symptomatic ( P40 ) ICV + IV injection (n=5), but not ICV only (n=2). Wild-type (n=5); Pla2g6 KO/G373R (n=10). Rotarod performance of mice with the indicated genotypes was measured weekly. ( B ) Pre-symptomatic ( P40 ) ICV + IV injection of AAV-PHP.eB-hPLA2G6 stabilizes body weight of Pla2g6 KO/G373R mice. Wild-type (n=5); Pla2g6 KO/G373R (n=6); Pla2g6 KO/G373R injected (n=3). ( C ) Pre-symptomatic ( P40 ) ICV + IV injection of AAV-PHP.eB-PLA2G6 prolongs lifespan of the Pla2g6 KO/G373R mice. Pla2g6 KO/G373R (n=8); Pla2g6 KO/G373R injected (n=8). Error bars represent SEM. * P<0.05; ** P<0.01; *** P<0.001. All assays were conducted in blind of genotypes and treatments.

Techniques: IV Injection, Injection

( A ) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. GlcCer antibody (green; arrows) was used to assess the levels of GlcCer. Scale bar=100 µm. All assays were conducted blind for genotypes and treatments. ( B ) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. LAMP2 antibody (green; arrows) labels lysosomes in the Purkinje cells. Scale bar=20 µm. ( C ) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. Vps35 antibody (green; arrows). Scale bar=20 µm. Representative images are shown in this figure. DAPI (blue) labels nuclei. Representative images are shown in this figure. Quantifications are shown next to the images (n=3 for wild-type and Pla2g6 KO/G373R mice; n=2 for Pla2g6 KO/G373R -injected mice). Error bars represent SEM; *p<0.05; **p<0.01. ICV, intracerebroventricular; IV, intravenous.

Journal: eLife

Article Title: Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)

doi: 10.7554/eLife.82555

Figure Lengend Snippet: ( A ) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. GlcCer antibody (green; arrows) was used to assess the levels of GlcCer. Scale bar=100 µm. All assays were conducted blind for genotypes and treatments. ( B ) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. LAMP2 antibody (green; arrows) labels lysosomes in the Purkinje cells. Scale bar=20 µm. ( C ) Immunofluorescent staining of mouse cerebella of the indicated genotypes and treatments. Vps35 antibody (green; arrows). Scale bar=20 µm. Representative images are shown in this figure. DAPI (blue) labels nuclei. Representative images are shown in this figure. Quantifications are shown next to the images (n=3 for wild-type and Pla2g6 KO/G373R mice; n=2 for Pla2g6 KO/G373R -injected mice). Error bars represent SEM; *p<0.05; **p<0.01. ICV, intracerebroventricular; IV, intravenous.

Techniques: Staining, Injection

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