mouse anti dok7 antibody  (Santa Cruz Biotechnology)

Journal: Brain Communications

Article Title: Dose escalation pre-clinical trial of novel DOK7-AAV in mouse model of DOK7 congenital myasthenia

doi: 10.1093/braincomms/fcaf046

Figure Lengend Snippet: DOK7 is expressed in diaphragm, triceps brachii and TA in AMP-101-treated Dok-7 KI/KI model mice. ( A ) Examples of western blots showing DOK7 expression. Robust expression is seen in muscles from many of the treated mice. Endogenous levels of DOK7 in WT mice are very low. ( B ) Densitometry was carried out to semi-quantify DOK7 expression. First, the ratio of the band intensity of DOK7 with total protein was calculated, and then this was normalized with a positive control (lysate from HEK292T cells transfected with human DOK7 cDNA). Each point represents a mouse, n = 4–5. P -values were obtained using a one-way ANOVA with Tukey’s multiple comparison test. Bars show mean values.

Article Snippet: Nitrocellulose was incubated in blocking buffer (PBS containing 3% powdered skimmed milk) for 30 min at RT and then incubated with primary mouse anti-DOK7 antibody (Santa Cruz, cat. no. sc-390856) diluted 1:250 in blocking buffer for 1 h at RT with gentle agitation.

Techniques: Western Blot, Expressing, Muscles, Positive Control, Transfection, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Long-term muscle-specific overexpression of DOK7 in mice using AAV9-tMCK-DOK7

doi: 10.1016/j.omtn.2023.07.036

Figure Lengend Snippet: AAV9-tMCK-DOK7 increases DOK7 expression in skeletal muscle (A) Schematic of the AAV9-tMCK-DOK7 plasmid showing the skeletal muscle targeting tMCK promoter and DOK7 transgene. (B–D) DOK7 expression across a range of muscles and organs from 1-month-old mice treated with high dose (5e11 vg), low dose (1.25e11 vg), or untreated with AAV9-tMCK-DOK7. “IS” denotes internal standards run in triplicate used to allow comparison between blots. (B) Western blots where the molecular weight of DOK7 is indicated. Dotted lines illustrate where blot sections have been cropped together. (C) Bar graph of endogenous expression in skeletal muscle and tissue shown by quantified western blots. Values are normalized to the average of three DOK7 internal standards (ISs). Each dot represents a single mouse, and bars represent the mean (with SEM) (n = 3 per bar). (D) DOK7 expression in response to high-dose or low-dose treatments were compared by one-way ANOVA with Holm-Šídák’s multiple comparisons. Each bar represents an average of three animals (with SEM), normalized to internal standard (IS) and then normalized to endogenous protein levels for that tissue. Expression increased by 40x over endogenous levels in the GC (p = 0.0114), and the TA (p = 0.0242), hindlimb lumbricals (p = 0.0074), and TVA (p = 0.013) each showed a marked rise in DOK7. The soleus, which exhibited the highest endogenous levels of DOK7 in skeletal muscle, still expressed ∼4x as much DOK7 following high-dose treatment (p = 0.0032). There was also a small but significant increase in DOK7 expression in the heart following high-dose AAV9-tMCK-DOK7 treatment (p = 0.0018, n = 3 in all groups, one-way ANOVAs with Holm-Šídák’s comparisons). SPC, spinal cord; TVA, transverse abdominis; TA, tibialis anterior; GC, gastrocnemius; ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: After gel electrophoresis, the protein was transferred to a polyvinylidene fluoride and the membranes was stained and imaged for total protein (Revert 700 Total protein stain, TPS; Li-COR 926–11010)., Blocking in Intercept PBS blocking buffer (Li-COR 927–70001) preceded 4°C overnight incubation in goat anti-DOK7 primary antibody (Cat: AF6398, R&D) , , at 1:1,500 in blocking buffer.

Techniques: Expressing, Plasmid Preparation, Muscles, Comparison, Western Blot, Molecular Weight

Journal: Molecular Therapy. Nucleic Acids

Article Title: Long-term muscle-specific overexpression of DOK7 in mice using AAV9-tMCK-DOK7

doi: 10.1016/j.omtn.2023.07.036

Figure Lengend Snippet: hDOK7 overexpression stimulates long-term elevation of DOK7 protein expression in mouse hindlimb muscles but not the heart (A) Western blots of tissue lysates probed with anti-DOK7 antibody shown at 55 kDa (middle band). Tissue from three mice each untreated or injected with high- or low-dose AAV9-tMCK-DOK7 was run alongside internal standards (ISs) produced in triplicate, enabling cross-gel comparison. Tissues were taken from mice at 3 months (M) and 6 months of age. (B–D) Bar charts comparing the effect of dose on tissue-specific DOK7 expression. Western blot data were quantified and normalized to the average value of three internal standards. DOK7 expression was significantly increased at 3 and 6 months following high-dose treatment in the hindlimb soleus muscle (B) and lumbricals (C) but not in heart tissue (D), when compared to untreated controls. Specifically, there was a 3.8-fold increase in DOK7 expression in the soleus 3 months following treatment and a 1.9-fold increase at 6 months (B; p < 0.0001 and p = 0.019), while in the lumbricals, there was a 4.7-fold increase in DOK7 expression at 3 months and a 4.6-fold increase at 6 months (C; p = 0.0009 and p = 0.0031) (one-way ANOVAs with Šídák’s multiple comparisons, means of n = 3 per group [with SEM], and each data point represents one animal). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: After gel electrophoresis, the protein was transferred to a polyvinylidene fluoride and the membranes was stained and imaged for total protein (Revert 700 Total protein stain, TPS; Li-COR 926–11010)., Blocking in Intercept PBS blocking buffer (Li-COR 927–70001) preceded 4°C overnight incubation in goat anti-DOK7 primary antibody (Cat: AF6398, R&D) , , at 1:1,500 in blocking buffer.

Techniques: Over Expression, Expressing, Muscles, Western Blot, Injection, Produced, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Long-term muscle-specific overexpression of DOK7 in mice using AAV9-tMCK-DOK7

doi: 10.1016/j.omtn.2023.07.036

Figure Lengend Snippet: Overexpression of DOK7 in skeletal muscle does not affect animal wellbeing nor motor development (A) Righting reflex measured from P1 to P13 when all animals consistently achieved the minimum time to right (1 s). Each point represents the average time of approximately 30 animals per group (mean [SEM]). Comparing between AAV9-tMCK-DOK7 doses revealed no difference among high-dose or low-dose treated or untreated animals (ns, n = 88; two-way ANOVA with Tukey’s post hoc comparisons). (B) Body weights (g) of mice from day 1 (day of birth) to 6 months (experimental endpoint). Dotted lines indicate where at day 30 and day 90, mice from each group were sacrificed for tissue, making n per group = 30, 20, and 10 from day 0, day 30, and day 90, respectively (mean and SEM). There was no difference observed between AAV9-tMCK-DOK7 doses (two-way ANOVA with Tukey’s multiple comparisons). (C–I) Plasma biochemistry measuring biomarkers from high- or low-dose-treated animals or untreated controls at 3 or 6 months old. Each data point represents a readout from one mouse, and bars represent averages from three mice (with SEM, one-way ANOVAs). ALT, alanine transaminase; AST, aspartate transaminase; ns, not significant.

Article Snippet: After gel electrophoresis, the protein was transferred to a polyvinylidene fluoride and the membranes was stained and imaged for total protein (Revert 700 Total protein stain, TPS; Li-COR 926–11010)., Blocking in Intercept PBS blocking buffer (Li-COR 927–70001) preceded 4°C overnight incubation in goat anti-DOK7 primary antibody (Cat: AF6398, R&D) , , at 1:1,500 in blocking buffer.

Techniques: Over Expression, Clinical Proteomics

Journal: Molecular Therapy. Nucleic Acids

Article Title: Long-term muscle-specific overexpression of DOK7 in mice using AAV9-tMCK-DOK7

doi: 10.1016/j.omtn.2023.07.036

Figure Lengend Snippet: Overexpression of DOK7 in skeletal muscle does not damage myofibers (A) Micrographs of H&E-stained soleus muscles from 6-month-old animals showing (A) minimal myofiber degeneration in a male treated with high-dose smAAV9- DOK7 highlighted with a black arrow, 40x. (B) Skeletal muscle regeneration, characterized by rows of plump nuclei in the central myofiber, in the soleus of a low-dose-treated animal, highlighted by green arrows, 20x. (C) Normal presentation in an untreated male, 20x. (D) Focal myofiber degeneration highlighted by the black arrow in the soleus of an untreated animal. For further histology results, see Tables S1–S3 .

Article Snippet: After gel electrophoresis, the protein was transferred to a polyvinylidene fluoride and the membranes was stained and imaged for total protein (Revert 700 Total protein stain, TPS; Li-COR 926–11010)., Blocking in Intercept PBS blocking buffer (Li-COR 927–70001) preceded 4°C overnight incubation in goat anti-DOK7 primary antibody (Cat: AF6398, R&D) , , at 1:1,500 in blocking buffer.

Techniques: Over Expression, Staining, Muscles

Journal: Molecular Therapy. Nucleic Acids

Article Title: Long-term muscle-specific overexpression of DOK7 in mice using AAV9-tMCK-DOK7

doi: 10.1016/j.omtn.2023.07.036

Figure Lengend Snippet: Innervation of hindlimb lumbrical muscle NMJs is not affected by AAV9-tMCK-DOK7 overexpression (A and B) NMJs labeled with α-bungarotoxin (AChRs) in magenta and anti-synaptic vesicle 2 (SV2) and anti-2H3 (neurofilaments) in green are shown in representative confocal micrographs. High-dose and low-dose AAV9-MCK-DOK7-treated and untreated lumbricals were taken at 3 months (A) and 6 months (B). Yellow arrows indicate partially denervated NMJs. Scale bars (white), 20 μm. (C and E) Percentages of NMJs fully innervated, partially innervated, or denervated presented by AAV9-tMCK-DOK7 dose condition. Mean percentages were computed from counts from three mice per condition (mean [SEM]). A minimum of 28 NMJs per muscle from each mouse were evaluated in tissue from 3- (C) and 6- (E) month-old mice. (D and F) Bar charts comparing the percentage of fully innervated NMJs between treatment conditions, showing results from the lumbricals taken from mice at 3 (D) and 6 (F) months old. Each point represents data from one animal and each bar represents an average of three animals with SEM. There was no difference between treatments as assessed a Kruskal-Wallis test with Dunn’s multiple comparisons (ns) (total number of NMJs analyzed per condition: untreated: 3M lumbricals, n = 232; 6M lumbricals, n = 190; low dose: 3M lumbricals, n = 129; 6M lumbricals, n = 165; high dose: 3M lumbricals, n = 222; 6M lumbricals, n = 134).

Article Snippet: After gel electrophoresis, the protein was transferred to a polyvinylidene fluoride and the membranes was stained and imaged for total protein (Revert 700 Total protein stain, TPS; Li-COR 926–11010)., Blocking in Intercept PBS blocking buffer (Li-COR 927–70001) preceded 4°C overnight incubation in goat anti-DOK7 primary antibody (Cat: AF6398, R&D) , , at 1:1,500 in blocking buffer.

Techniques: Over Expression, Labeling

Journal: Molecular Therapy. Nucleic Acids

Article Title: Long-term muscle-specific overexpression of DOK7 in mice using AAV9-tMCK-DOK7

doi: 10.1016/j.omtn.2023.07.036

Figure Lengend Snippet: Long-term enlargement of lumbrical NMJs after AAV9-tMCK-DOK7 treatment (A–L) Bar charts comparing NMJ morphology in mice injected with low- or high-dose AAV9-tMCK-DOK7 , 3 or 6 months prior. One data point represents the average of a minimum of 20 NMJs from the hindlimb lumbrical muscles of a single mouse. Each bar represents the mean (SEM) (n = 3 per group). At 6 months post injection, there was a significant increase in endplate area in low- (J; p = 0.011) and high-dose (L; p = 0.0006) -treated animals, with an over 100% increase following the high dose. Nerve terminal area was also increased 6 months after low- (F; p = 0.042) and high-dose (H; p = 0.0007) treatment, as assessed using unpaired t tests. Untreated groups were repeatedly used in comparison with low- or high-dosed muscle samples using unpaired t tests for comparisons. (M and N) Confocal micrographs of NMJs labeled with α-bungarotoxin (AChRs) in magenta and anti-synaptic vesicle 2 (SV2) and anti-2H3 (neurofilaments) in green. Images are representative of lumbrical muscles taken from 6-month-old mice treated with high-dose AAV9-tMCK-DOK7 (N) or untreated (M) at P1. ∗p < 0.05, p∗∗∗ < 0.001.

Article Snippet: After gel electrophoresis, the protein was transferred to a polyvinylidene fluoride and the membranes was stained and imaged for total protein (Revert 700 Total protein stain, TPS; Li-COR 926–11010)., Blocking in Intercept PBS blocking buffer (Li-COR 927–70001) preceded 4°C overnight incubation in goat anti-DOK7 primary antibody (Cat: AF6398, R&D) , , at 1:1,500 in blocking buffer.

Techniques: Injection, Muscles, Comparison, Labeling