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    OriGene dnm1l
    Longitudinal IOP monitoring. Rebound tonometry was used to examine IOP once a week. IOP was stably elevated three weeks after episcleral vein occlusion (EVO) at the OS. Stable IOP elevation was followed by intravitreal injection (IVI) of either IgG control antibody (IgG OS, Black) or <t>anti-DNM1L</t> antibody (DNM1L OS, Red). The contralateral OD, IgG OD (light gray), and DNM1L OD (dark gray), received no interventions and served as internal controls. IOP remained stably elevated until the end of the study. The increase in w10 was statistically significant. **** p < 0.0001, student’s t -test. Numbers of animals used: n = 7 (IgG), n = 8 (DNM1L).
    Dnm1l, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ap55855pu+s/DRP1+(DNM1L)+pSer637+Rabbit+Polyclonal+Antibody/pmc09654827-21-0-10
    Average 90 stars, based on 1 article reviews
    dnm1l - by Bioz Stars, 2026-11
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    1) Product Images from "Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma"

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms232113618

    Longitudinal IOP monitoring. Rebound tonometry was used to examine IOP once a week. IOP was stably elevated three weeks after episcleral vein occlusion (EVO) at the OS. Stable IOP elevation was followed by intravitreal injection (IVI) of either IgG control antibody (IgG OS, Black) or anti-DNM1L antibody (DNM1L OS, Red). The contralateral OD, IgG OD (light gray), and DNM1L OD (dark gray), received no interventions and served as internal controls. IOP remained stably elevated until the end of the study. The increase in w10 was statistically significant. **** p < 0.0001, student’s t -test. Numbers of animals used: n = 7 (IgG), n = 8 (DNM1L).
    Figure Legend Snippet: Longitudinal IOP monitoring. Rebound tonometry was used to examine IOP once a week. IOP was stably elevated three weeks after episcleral vein occlusion (EVO) at the OS. Stable IOP elevation was followed by intravitreal injection (IVI) of either IgG control antibody (IgG OS, Black) or anti-DNM1L antibody (DNM1L OS, Red). The contralateral OD, IgG OD (light gray), and DNM1L OD (dark gray), received no interventions and served as internal controls. IOP remained stably elevated until the end of the study. The increase in w10 was statistically significant. **** p < 0.0001, student’s t -test. Numbers of animals used: n = 7 (IgG), n = 8 (DNM1L).

    Techniques Used: Stable Transfection, Injection

    Immunofluorescence staining of RGCs with Brn3a. ( A ) Example of a retinal flatmount with RGCs stained by indirect immunofluorescence of Brn3a. The flatmount is divided into three sections: central (1), mid-periphery (2), and periphery (3). Scale bar: 1 mm. Three images are recorded within each region. Calculating the RGC count was performed by summing up the individual count of each image and then dividing by the number of images included. For regional analysis of the RGC count, the RGC count of three images of each region is summed up and divided by the number of images used for calculation. ( B ) Representative Brn3a stained images of each region. IgG OS group (left panel), DNM1L OS group (right panel). Scale bar: 50 µm.
    Figure Legend Snippet: Immunofluorescence staining of RGCs with Brn3a. ( A ) Example of a retinal flatmount with RGCs stained by indirect immunofluorescence of Brn3a. The flatmount is divided into three sections: central (1), mid-periphery (2), and periphery (3). Scale bar: 1 mm. Three images are recorded within each region. Calculating the RGC count was performed by summing up the individual count of each image and then dividing by the number of images included. For regional analysis of the RGC count, the RGC count of three images of each region is summed up and divided by the number of images used for calculation. ( B ) Representative Brn3a stained images of each region. IgG OS group (left panel), DNM1L OS group (right panel). Scale bar: 50 µm.

    Techniques Used: Immunofluorescence, Staining

    Quantitative analysis of Brn3 + RGCs in retinal flatmounts. Retinal ganglion cells were stained using immunostaining of Brn3a. An anti-Brn3a antibody (C20, Santa Cruz, Dallas, TX, USA) was used as the primary antibody and an anti-goat AF568 antibody (Life Technologies, Carlsbad, CA, USA) as the secondary antibody. Single images were taken with a fluorescence microscope (Eclipse TS 100 microscope, Nikon, Yurakucho, Tokyo, Japan), DS-Fi1-U2 digital microscope camera (Nikon, Tokyo, Japan). Objective: ELWD 20×/0.45 S Plan Flour Ph1 ADM Air objective (Nikon, Tokyo, Japan). ( A ) Nine images were taken from each flatmount. The RGC number was determined for each image. The average RGC number of the whole flatmount was calculated over all nine images. ( B ) Three of the total nine images were taken for each of the areas central, mid-peripheral, peripheral. For each area, the average RGC density was calculated over the three recordings of one area. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, One-way ANOVA, Tukey’s HSD test (post hoc). Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.
    Figure Legend Snippet: Quantitative analysis of Brn3 + RGCs in retinal flatmounts. Retinal ganglion cells were stained using immunostaining of Brn3a. An anti-Brn3a antibody (C20, Santa Cruz, Dallas, TX, USA) was used as the primary antibody and an anti-goat AF568 antibody (Life Technologies, Carlsbad, CA, USA) as the secondary antibody. Single images were taken with a fluorescence microscope (Eclipse TS 100 microscope, Nikon, Yurakucho, Tokyo, Japan), DS-Fi1-U2 digital microscope camera (Nikon, Tokyo, Japan). Objective: ELWD 20×/0.45 S Plan Flour Ph1 ADM Air objective (Nikon, Tokyo, Japan). ( A ) Nine images were taken from each flatmount. The RGC number was determined for each image. The average RGC number of the whole flatmount was calculated over all nine images. ( B ) Three of the total nine images were taken for each of the areas central, mid-peripheral, peripheral. For each area, the average RGC density was calculated over the three recordings of one area. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, One-way ANOVA, Tukey’s HSD test (post hoc). Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.

    Techniques Used: Staining, Immunostaining, Fluorescence, Microscopy

    Retinal nerve fiber layer (RNFL) thickness analysis. Optical coherence tomography (OCT) was used to scan the retinal cross-sections in vivo. OCT allowed the acquisition of different retinal layers. For the analysis of the acquired circular B-scans with 12° diameter, a semi-automatic algorithm was used to define the RNFL and calculate its thickness (RNFLT). The Heidelberg Eye Explorer software divided the scan into the main sectors superior (S), temporal (T), inferior (I), and nasal (N). Furthermore, the sectors temporal superior (TS), temporal inferior (TI), nasal inferior (NI), and nasal superior (NS) were included ( A ). In addition to the regional classification, the schematic illustration also showed the sides of the interventions, episcleral vein occlusion (EVO) and intravitreal injection (IVI), which were performed on the eye during the study (Scheme modified from . The mean RNFLT of the whole B-scan was used for follow-up quantification ( B ). Further quantification of the RNFLT at week 10 was performed for the individual sectors ( C – H ). Legend: black (IgG OS), red (DNM1L), light grey (IgG OD), dark grey (IgG OD), ** p < 0.01, *** p < 0.001, ns = not significant; One-way ANOVA, Tukey’s HSD test (post hoc). Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.
    Figure Legend Snippet: Retinal nerve fiber layer (RNFL) thickness analysis. Optical coherence tomography (OCT) was used to scan the retinal cross-sections in vivo. OCT allowed the acquisition of different retinal layers. For the analysis of the acquired circular B-scans with 12° diameter, a semi-automatic algorithm was used to define the RNFL and calculate its thickness (RNFLT). The Heidelberg Eye Explorer software divided the scan into the main sectors superior (S), temporal (T), inferior (I), and nasal (N). Furthermore, the sectors temporal superior (TS), temporal inferior (TI), nasal inferior (NI), and nasal superior (NS) were included ( A ). In addition to the regional classification, the schematic illustration also showed the sides of the interventions, episcleral vein occlusion (EVO) and intravitreal injection (IVI), which were performed on the eye during the study (Scheme modified from . The mean RNFLT of the whole B-scan was used for follow-up quantification ( B ). Further quantification of the RNFLT at week 10 was performed for the individual sectors ( C – H ). Legend: black (IgG OS), red (DNM1L), light grey (IgG OD), dark grey (IgG OD), ** p < 0.01, *** p < 0.001, ns = not significant; One-way ANOVA, Tukey’s HSD test (post hoc). Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.

    Techniques Used: Tomography, In Vivo, Software, Injection, Modification

    Analysis of retinal functionality using photopic Ganzfeld ERG. To determine retinal functionality, photopic Ganzfeld ERGs of a protocol with increasing light flash intensity were acquired. ERG recordings at baseline (w0, before episcleral vein occlusion, EVO) and at the final examination at w10 after EVO were arithmetically averaged. For the ERG recordings and quantification shown, the flash intensity 1.37 log 10 cd·s·m −2 was used. Thereby, the ERG profile for the IgG group in w0 ( A ) showed no difference between the left (OS) and right (OD) eyes. For quantification, the amplitudes of the profiles, B-wave (*), and photopic negative response (PhNR, **) were used. ( B ) The ERG profile of the IgG group at w10 showed a decrease in B-wave and PhNR amplitudes. ( C ) For quantification in the IgG group, B-wave amplitude ( C ) and PhNR amplitude ( D ) were analyzed. For the DNM1L group, ERG profiles were acquired at time point w0 ( E ) and time point w10 ( F ). B-wave amplitude ( G ) and PhNR amplitude ( H ) were analyzed for w0 and w10. The millivolt decrease was calculated as percent difference (Δ = % change). Number of animals used: n = 7 (IgG), n = 8 (DNM1L).
    Figure Legend Snippet: Analysis of retinal functionality using photopic Ganzfeld ERG. To determine retinal functionality, photopic Ganzfeld ERGs of a protocol with increasing light flash intensity were acquired. ERG recordings at baseline (w0, before episcleral vein occlusion, EVO) and at the final examination at w10 after EVO were arithmetically averaged. For the ERG recordings and quantification shown, the flash intensity 1.37 log 10 cd·s·m −2 was used. Thereby, the ERG profile for the IgG group in w0 ( A ) showed no difference between the left (OS) and right (OD) eyes. For quantification, the amplitudes of the profiles, B-wave (*), and photopic negative response (PhNR, **) were used. ( B ) The ERG profile of the IgG group at w10 showed a decrease in B-wave and PhNR amplitudes. ( C ) For quantification in the IgG group, B-wave amplitude ( C ) and PhNR amplitude ( D ) were analyzed. For the DNM1L group, ERG profiles were acquired at time point w0 ( E ) and time point w10 ( F ). B-wave amplitude ( G ) and PhNR amplitude ( H ) were analyzed for w0 and w10. The millivolt decrease was calculated as percent difference (Δ = % change). Number of animals used: n = 7 (IgG), n = 8 (DNM1L).

    Techniques Used:

    Anti-DNM1L Ab injection alters the phosphorylation of DNM1L. ( A ) Western blot detection of DNM1L, total protein expression (DNM1L total ), phosphorylation of DNM1L at serine 635 (DNM1L p635 ), phosphorylation of DNM1L as serine 656 (DNM1L p656 ), and housekeeping protein beta-actin. The presented representative western blot is one of six biological replicates. Densitometric analysis of DNM1L total ( B ). All calculated values were normalized to beta-actin. The resulting fold-change was subsequently normalized to the total DNM1L protein expression: ( C ) DNM1L p635/total ), ( D ) DNM1L p656/total . ( E ) The ratio of the DNM1L phosphorylation sites p635 and p656 was calculated (DNM1L p635 /DNM1L p656 ). All values are given as mean ± SEM calculated from six biological replicates. Statistical analysis was performed using student’s t -test. Significance level: * = p < 0.05, ** = p < 0.01, ns = not significant. Numbers of animals used: n = 6 (IgG), n = 6 DNM1L.
    Figure Legend Snippet: Anti-DNM1L Ab injection alters the phosphorylation of DNM1L. ( A ) Western blot detection of DNM1L, total protein expression (DNM1L total ), phosphorylation of DNM1L at serine 635 (DNM1L p635 ), phosphorylation of DNM1L as serine 656 (DNM1L p656 ), and housekeeping protein beta-actin. The presented representative western blot is one of six biological replicates. Densitometric analysis of DNM1L total ( B ). All calculated values were normalized to beta-actin. The resulting fold-change was subsequently normalized to the total DNM1L protein expression: ( C ) DNM1L p635/total ), ( D ) DNM1L p656/total . ( E ) The ratio of the DNM1L phosphorylation sites p635 and p656 was calculated (DNM1L p635 /DNM1L p656 ). All values are given as mean ± SEM calculated from six biological replicates. Statistical analysis was performed using student’s t -test. Significance level: * = p < 0.05, ** = p < 0.01, ns = not significant. Numbers of animals used: n = 6 (IgG), n = 6 DNM1L.

    Techniques Used: Injection, Western Blot, Expressing

    Anti-DNM1L Ab injection reduces proteins involved in mitochondrial apoptosis. ( A ) Western blot detection of XIAP, total protein expression of BAD (BAD total ), phosphorylation of BAD at serine 112 (BAD p112 ), and housekeeping protein beta-actin. The presented representative western blot is one of six biological replicates. Densitometric analysis of XIAP ( B ), BAD total ( C ), and BAD p112 ( D ). All calculated values were normalized to beta-actin. The ratio of BAD p112 /BAD total was calculated ( E ). All values are given as mean ± SEM calculated from six biological replicates. Statistical analysis was performed using student’s t -test. Significance level: * = p < 0.05, ** = p < 0.01, ns = not significant. Numbers of animals used: n = 6 (IgG), n = 6 DNM1L.
    Figure Legend Snippet: Anti-DNM1L Ab injection reduces proteins involved in mitochondrial apoptosis. ( A ) Western blot detection of XIAP, total protein expression of BAD (BAD total ), phosphorylation of BAD at serine 112 (BAD p112 ), and housekeeping protein beta-actin. The presented representative western blot is one of six biological replicates. Densitometric analysis of XIAP ( B ), BAD total ( C ), and BAD p112 ( D ). All calculated values were normalized to beta-actin. The ratio of BAD p112 /BAD total was calculated ( E ). All values are given as mean ± SEM calculated from six biological replicates. Statistical analysis was performed using student’s t -test. Significance level: * = p < 0.05, ** = p < 0.01, ns = not significant. Numbers of animals used: n = 6 (IgG), n = 6 DNM1L.

    Techniques Used: Injection, Western Blot, Expressing

    Illustration of the up- and down-regulation of significantly altered protein expression identified by mass spectrometric analysis. After sacrifice of the animals at the end of the study, the proteins were isolated from retinal tissue and tryptically digested. The resulting peptides were purified and analyzed by MS. Identification, quantification and normalization of the peptides was performed using MaxQuant. For statistical analysis, a Student’s t -test was used with a significance level p < 0.05. The graph shows the up—( A ) and down-regulated ( B ) proteins in the antibody-treated group compared to the control group. Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.
    Figure Legend Snippet: Illustration of the up- and down-regulation of significantly altered protein expression identified by mass spectrometric analysis. After sacrifice of the animals at the end of the study, the proteins were isolated from retinal tissue and tryptically digested. The resulting peptides were purified and analyzed by MS. Identification, quantification and normalization of the peptides was performed using MaxQuant. For statistical analysis, a Student’s t -test was used with a significance level p < 0.05. The graph shows the up—( A ) and down-regulated ( B ) proteins in the antibody-treated group compared to the control group. Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.

    Techniques Used: Expressing, Isolation, Purification

    STRING protein interaction network. Analysis of the signaling pathway altered by the anti-DNM1L Ab injection. Search tool for the retrieval of interacting proteins (STRING) shows the signaling pathways of the most significantly changed proteins using the medium confidence score (0.4). The target protein of the study, DNM1L, was added manually to the network.
    Figure Legend Snippet: STRING protein interaction network. Analysis of the signaling pathway altered by the anti-DNM1L Ab injection. Search tool for the retrieval of interacting proteins (STRING) shows the signaling pathways of the most significantly changed proteins using the medium confidence score (0.4). The target protein of the study, DNM1L, was added manually to the network.

    Techniques Used: Injection

    MS identified proteins, categorized by subcellular location or association.
    Figure Legend Snippet: MS identified proteins, categorized by subcellular location or association.

    Techniques Used:

    List of antibodies used for western blot. The antibodies were diluted, if not stated differently, in 5% BSA and 0.1% NaN3 in TBS-T. Abbreviations: rb = rabbit, ms = mouse. * The phosphorylation sites in the human DNM1L are at positions Ser616 and Ser637. Due to a 100% sequence homology in the BLAST comparison of human with rat DNM1L, we assume that the antibody used also detect the rat-specific phosphorylation sites at Ser635 (Ser616, human) or Ser656 (Ser637, human). Since rat tissue was used in this manuscript, we termed the DNM1L phosphorylation sites according to the rat-specific positions.
    Figure Legend Snippet: List of antibodies used for western blot. The antibodies were diluted, if not stated differently, in 5% BSA and 0.1% NaN3 in TBS-T. Abbreviations: rb = rabbit, ms = mouse. * The phosphorylation sites in the human DNM1L are at positions Ser616 and Ser637. Due to a 100% sequence homology in the BLAST comparison of human with rat DNM1L, we assume that the antibody used also detect the rat-specific phosphorylation sites at Ser635 (Ser616, human) or Ser656 (Ser637, human). Since rat tissue was used in this manuscript, we termed the DNM1L phosphorylation sites according to the rat-specific positions.

    Techniques Used: Western Blot, Sequencing

    Related Articles

    other:

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma
    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.



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    Longitudinal IOP monitoring. Rebound tonometry was used to examine IOP once a week. IOP was stably elevated three weeks after episcleral vein occlusion (EVO) at the OS. Stable IOP elevation was followed by intravitreal injection (IVI) of either IgG control antibody (IgG OS, Black) or <t>anti-DNM1L</t> antibody (DNM1L OS, Red). The contralateral OD, IgG OD (light gray), and DNM1L OD (dark gray), received no interventions and served as internal controls. IOP remained stably elevated until the end of the study. The increase in w10 was statistically significant. **** p < 0.0001, student’s t -test. Numbers of animals used: n = 7 (IgG), n = 8 (DNM1L).
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    Image Search Results


    Longitudinal IOP monitoring. Rebound tonometry was used to examine IOP once a week. IOP was stably elevated three weeks after episcleral vein occlusion (EVO) at the OS. Stable IOP elevation was followed by intravitreal injection (IVI) of either IgG control antibody (IgG OS, Black) or anti-DNM1L antibody (DNM1L OS, Red). The contralateral OD, IgG OD (light gray), and DNM1L OD (dark gray), received no interventions and served as internal controls. IOP remained stably elevated until the end of the study. The increase in w10 was statistically significant. **** p < 0.0001, student’s t -test. Numbers of animals used: n = 7 (IgG), n = 8 (DNM1L).

    Journal: International Journal of Molecular Sciences

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    doi: 10.3390/ijms232113618

    Figure Lengend Snippet: Longitudinal IOP monitoring. Rebound tonometry was used to examine IOP once a week. IOP was stably elevated three weeks after episcleral vein occlusion (EVO) at the OS. Stable IOP elevation was followed by intravitreal injection (IVI) of either IgG control antibody (IgG OS, Black) or anti-DNM1L antibody (DNM1L OS, Red). The contralateral OD, IgG OD (light gray), and DNM1L OD (dark gray), received no interventions and served as internal controls. IOP remained stably elevated until the end of the study. The increase in w10 was statistically significant. **** p < 0.0001, student’s t -test. Numbers of animals used: n = 7 (IgG), n = 8 (DNM1L).

    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.

    Techniques: Stable Transfection, Injection

    Immunofluorescence staining of RGCs with Brn3a. ( A ) Example of a retinal flatmount with RGCs stained by indirect immunofluorescence of Brn3a. The flatmount is divided into three sections: central (1), mid-periphery (2), and periphery (3). Scale bar: 1 mm. Three images are recorded within each region. Calculating the RGC count was performed by summing up the individual count of each image and then dividing by the number of images included. For regional analysis of the RGC count, the RGC count of three images of each region is summed up and divided by the number of images used for calculation. ( B ) Representative Brn3a stained images of each region. IgG OS group (left panel), DNM1L OS group (right panel). Scale bar: 50 µm.

    Journal: International Journal of Molecular Sciences

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    doi: 10.3390/ijms232113618

    Figure Lengend Snippet: Immunofluorescence staining of RGCs with Brn3a. ( A ) Example of a retinal flatmount with RGCs stained by indirect immunofluorescence of Brn3a. The flatmount is divided into three sections: central (1), mid-periphery (2), and periphery (3). Scale bar: 1 mm. Three images are recorded within each region. Calculating the RGC count was performed by summing up the individual count of each image and then dividing by the number of images included. For regional analysis of the RGC count, the RGC count of three images of each region is summed up and divided by the number of images used for calculation. ( B ) Representative Brn3a stained images of each region. IgG OS group (left panel), DNM1L OS group (right panel). Scale bar: 50 µm.

    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.

    Techniques: Immunofluorescence, Staining

    Quantitative analysis of Brn3 + RGCs in retinal flatmounts. Retinal ganglion cells were stained using immunostaining of Brn3a. An anti-Brn3a antibody (C20, Santa Cruz, Dallas, TX, USA) was used as the primary antibody and an anti-goat AF568 antibody (Life Technologies, Carlsbad, CA, USA) as the secondary antibody. Single images were taken with a fluorescence microscope (Eclipse TS 100 microscope, Nikon, Yurakucho, Tokyo, Japan), DS-Fi1-U2 digital microscope camera (Nikon, Tokyo, Japan). Objective: ELWD 20×/0.45 S Plan Flour Ph1 ADM Air objective (Nikon, Tokyo, Japan). ( A ) Nine images were taken from each flatmount. The RGC number was determined for each image. The average RGC number of the whole flatmount was calculated over all nine images. ( B ) Three of the total nine images were taken for each of the areas central, mid-peripheral, peripheral. For each area, the average RGC density was calculated over the three recordings of one area. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, One-way ANOVA, Tukey’s HSD test (post hoc). Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.

    Journal: International Journal of Molecular Sciences

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    doi: 10.3390/ijms232113618

    Figure Lengend Snippet: Quantitative analysis of Brn3 + RGCs in retinal flatmounts. Retinal ganglion cells were stained using immunostaining of Brn3a. An anti-Brn3a antibody (C20, Santa Cruz, Dallas, TX, USA) was used as the primary antibody and an anti-goat AF568 antibody (Life Technologies, Carlsbad, CA, USA) as the secondary antibody. Single images were taken with a fluorescence microscope (Eclipse TS 100 microscope, Nikon, Yurakucho, Tokyo, Japan), DS-Fi1-U2 digital microscope camera (Nikon, Tokyo, Japan). Objective: ELWD 20×/0.45 S Plan Flour Ph1 ADM Air objective (Nikon, Tokyo, Japan). ( A ) Nine images were taken from each flatmount. The RGC number was determined for each image. The average RGC number of the whole flatmount was calculated over all nine images. ( B ) Three of the total nine images were taken for each of the areas central, mid-peripheral, peripheral. For each area, the average RGC density was calculated over the three recordings of one area. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant, One-way ANOVA, Tukey’s HSD test (post hoc). Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.

    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.

    Techniques: Staining, Immunostaining, Fluorescence, Microscopy

    Retinal nerve fiber layer (RNFL) thickness analysis. Optical coherence tomography (OCT) was used to scan the retinal cross-sections in vivo. OCT allowed the acquisition of different retinal layers. For the analysis of the acquired circular B-scans with 12° diameter, a semi-automatic algorithm was used to define the RNFL and calculate its thickness (RNFLT). The Heidelberg Eye Explorer software divided the scan into the main sectors superior (S), temporal (T), inferior (I), and nasal (N). Furthermore, the sectors temporal superior (TS), temporal inferior (TI), nasal inferior (NI), and nasal superior (NS) were included ( A ). In addition to the regional classification, the schematic illustration also showed the sides of the interventions, episcleral vein occlusion (EVO) and intravitreal injection (IVI), which were performed on the eye during the study (Scheme modified from . The mean RNFLT of the whole B-scan was used for follow-up quantification ( B ). Further quantification of the RNFLT at week 10 was performed for the individual sectors ( C – H ). Legend: black (IgG OS), red (DNM1L), light grey (IgG OD), dark grey (IgG OD), ** p < 0.01, *** p < 0.001, ns = not significant; One-way ANOVA, Tukey’s HSD test (post hoc). Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.

    Journal: International Journal of Molecular Sciences

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    doi: 10.3390/ijms232113618

    Figure Lengend Snippet: Retinal nerve fiber layer (RNFL) thickness analysis. Optical coherence tomography (OCT) was used to scan the retinal cross-sections in vivo. OCT allowed the acquisition of different retinal layers. For the analysis of the acquired circular B-scans with 12° diameter, a semi-automatic algorithm was used to define the RNFL and calculate its thickness (RNFLT). The Heidelberg Eye Explorer software divided the scan into the main sectors superior (S), temporal (T), inferior (I), and nasal (N). Furthermore, the sectors temporal superior (TS), temporal inferior (TI), nasal inferior (NI), and nasal superior (NS) were included ( A ). In addition to the regional classification, the schematic illustration also showed the sides of the interventions, episcleral vein occlusion (EVO) and intravitreal injection (IVI), which were performed on the eye during the study (Scheme modified from . The mean RNFLT of the whole B-scan was used for follow-up quantification ( B ). Further quantification of the RNFLT at week 10 was performed for the individual sectors ( C – H ). Legend: black (IgG OS), red (DNM1L), light grey (IgG OD), dark grey (IgG OD), ** p < 0.01, *** p < 0.001, ns = not significant; One-way ANOVA, Tukey’s HSD test (post hoc). Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.

    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.

    Techniques: Tomography, In Vivo, Software, Injection, Modification

    Analysis of retinal functionality using photopic Ganzfeld ERG. To determine retinal functionality, photopic Ganzfeld ERGs of a protocol with increasing light flash intensity were acquired. ERG recordings at baseline (w0, before episcleral vein occlusion, EVO) and at the final examination at w10 after EVO were arithmetically averaged. For the ERG recordings and quantification shown, the flash intensity 1.37 log 10 cd·s·m −2 was used. Thereby, the ERG profile for the IgG group in w0 ( A ) showed no difference between the left (OS) and right (OD) eyes. For quantification, the amplitudes of the profiles, B-wave (*), and photopic negative response (PhNR, **) were used. ( B ) The ERG profile of the IgG group at w10 showed a decrease in B-wave and PhNR amplitudes. ( C ) For quantification in the IgG group, B-wave amplitude ( C ) and PhNR amplitude ( D ) were analyzed. For the DNM1L group, ERG profiles were acquired at time point w0 ( E ) and time point w10 ( F ). B-wave amplitude ( G ) and PhNR amplitude ( H ) were analyzed for w0 and w10. The millivolt decrease was calculated as percent difference (Δ = % change). Number of animals used: n = 7 (IgG), n = 8 (DNM1L).

    Journal: International Journal of Molecular Sciences

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    doi: 10.3390/ijms232113618

    Figure Lengend Snippet: Analysis of retinal functionality using photopic Ganzfeld ERG. To determine retinal functionality, photopic Ganzfeld ERGs of a protocol with increasing light flash intensity were acquired. ERG recordings at baseline (w0, before episcleral vein occlusion, EVO) and at the final examination at w10 after EVO were arithmetically averaged. For the ERG recordings and quantification shown, the flash intensity 1.37 log 10 cd·s·m −2 was used. Thereby, the ERG profile for the IgG group in w0 ( A ) showed no difference between the left (OS) and right (OD) eyes. For quantification, the amplitudes of the profiles, B-wave (*), and photopic negative response (PhNR, **) were used. ( B ) The ERG profile of the IgG group at w10 showed a decrease in B-wave and PhNR amplitudes. ( C ) For quantification in the IgG group, B-wave amplitude ( C ) and PhNR amplitude ( D ) were analyzed. For the DNM1L group, ERG profiles were acquired at time point w0 ( E ) and time point w10 ( F ). B-wave amplitude ( G ) and PhNR amplitude ( H ) were analyzed for w0 and w10. The millivolt decrease was calculated as percent difference (Δ = % change). Number of animals used: n = 7 (IgG), n = 8 (DNM1L).

    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.

    Techniques:

    Anti-DNM1L Ab injection alters the phosphorylation of DNM1L. ( A ) Western blot detection of DNM1L, total protein expression (DNM1L total ), phosphorylation of DNM1L at serine 635 (DNM1L p635 ), phosphorylation of DNM1L as serine 656 (DNM1L p656 ), and housekeeping protein beta-actin. The presented representative western blot is one of six biological replicates. Densitometric analysis of DNM1L total ( B ). All calculated values were normalized to beta-actin. The resulting fold-change was subsequently normalized to the total DNM1L protein expression: ( C ) DNM1L p635/total ), ( D ) DNM1L p656/total . ( E ) The ratio of the DNM1L phosphorylation sites p635 and p656 was calculated (DNM1L p635 /DNM1L p656 ). All values are given as mean ± SEM calculated from six biological replicates. Statistical analysis was performed using student’s t -test. Significance level: * = p < 0.05, ** = p < 0.01, ns = not significant. Numbers of animals used: n = 6 (IgG), n = 6 DNM1L.

    Journal: International Journal of Molecular Sciences

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    doi: 10.3390/ijms232113618

    Figure Lengend Snippet: Anti-DNM1L Ab injection alters the phosphorylation of DNM1L. ( A ) Western blot detection of DNM1L, total protein expression (DNM1L total ), phosphorylation of DNM1L at serine 635 (DNM1L p635 ), phosphorylation of DNM1L as serine 656 (DNM1L p656 ), and housekeeping protein beta-actin. The presented representative western blot is one of six biological replicates. Densitometric analysis of DNM1L total ( B ). All calculated values were normalized to beta-actin. The resulting fold-change was subsequently normalized to the total DNM1L protein expression: ( C ) DNM1L p635/total ), ( D ) DNM1L p656/total . ( E ) The ratio of the DNM1L phosphorylation sites p635 and p656 was calculated (DNM1L p635 /DNM1L p656 ). All values are given as mean ± SEM calculated from six biological replicates. Statistical analysis was performed using student’s t -test. Significance level: * = p < 0.05, ** = p < 0.01, ns = not significant. Numbers of animals used: n = 6 (IgG), n = 6 DNM1L.

    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.

    Techniques: Injection, Western Blot, Expressing

    Anti-DNM1L Ab injection reduces proteins involved in mitochondrial apoptosis. ( A ) Western blot detection of XIAP, total protein expression of BAD (BAD total ), phosphorylation of BAD at serine 112 (BAD p112 ), and housekeeping protein beta-actin. The presented representative western blot is one of six biological replicates. Densitometric analysis of XIAP ( B ), BAD total ( C ), and BAD p112 ( D ). All calculated values were normalized to beta-actin. The ratio of BAD p112 /BAD total was calculated ( E ). All values are given as mean ± SEM calculated from six biological replicates. Statistical analysis was performed using student’s t -test. Significance level: * = p < 0.05, ** = p < 0.01, ns = not significant. Numbers of animals used: n = 6 (IgG), n = 6 DNM1L.

    Journal: International Journal of Molecular Sciences

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    doi: 10.3390/ijms232113618

    Figure Lengend Snippet: Anti-DNM1L Ab injection reduces proteins involved in mitochondrial apoptosis. ( A ) Western blot detection of XIAP, total protein expression of BAD (BAD total ), phosphorylation of BAD at serine 112 (BAD p112 ), and housekeeping protein beta-actin. The presented representative western blot is one of six biological replicates. Densitometric analysis of XIAP ( B ), BAD total ( C ), and BAD p112 ( D ). All calculated values were normalized to beta-actin. The ratio of BAD p112 /BAD total was calculated ( E ). All values are given as mean ± SEM calculated from six biological replicates. Statistical analysis was performed using student’s t -test. Significance level: * = p < 0.05, ** = p < 0.01, ns = not significant. Numbers of animals used: n = 6 (IgG), n = 6 DNM1L.

    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.

    Techniques: Injection, Western Blot, Expressing

    Illustration of the up- and down-regulation of significantly altered protein expression identified by mass spectrometric analysis. After sacrifice of the animals at the end of the study, the proteins were isolated from retinal tissue and tryptically digested. The resulting peptides were purified and analyzed by MS. Identification, quantification and normalization of the peptides was performed using MaxQuant. For statistical analysis, a Student’s t -test was used with a significance level p < 0.05. The graph shows the up—( A ) and down-regulated ( B ) proteins in the antibody-treated group compared to the control group. Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.

    Journal: International Journal of Molecular Sciences

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    doi: 10.3390/ijms232113618

    Figure Lengend Snippet: Illustration of the up- and down-regulation of significantly altered protein expression identified by mass spectrometric analysis. After sacrifice of the animals at the end of the study, the proteins were isolated from retinal tissue and tryptically digested. The resulting peptides were purified and analyzed by MS. Identification, quantification and normalization of the peptides was performed using MaxQuant. For statistical analysis, a Student’s t -test was used with a significance level p < 0.05. The graph shows the up—( A ) and down-regulated ( B ) proteins in the antibody-treated group compared to the control group. Numbers of animals used: n = 7 (IgG), n = 8 DNM1L.

    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.

    Techniques: Expressing, Isolation, Purification

    STRING protein interaction network. Analysis of the signaling pathway altered by the anti-DNM1L Ab injection. Search tool for the retrieval of interacting proteins (STRING) shows the signaling pathways of the most significantly changed proteins using the medium confidence score (0.4). The target protein of the study, DNM1L, was added manually to the network.

    Journal: International Journal of Molecular Sciences

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    doi: 10.3390/ijms232113618

    Figure Lengend Snippet: STRING protein interaction network. Analysis of the signaling pathway altered by the anti-DNM1L Ab injection. Search tool for the retrieval of interacting proteins (STRING) shows the signaling pathways of the most significantly changed proteins using the medium confidence score (0.4). The target protein of the study, DNM1L, was added manually to the network.

    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.

    Techniques: Injection

    MS identified proteins, categorized by subcellular location or association.

    Journal: International Journal of Molecular Sciences

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    doi: 10.3390/ijms232113618

    Figure Lengend Snippet: MS identified proteins, categorized by subcellular location or association.

    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.

    Techniques:

    List of antibodies used for western blot. The antibodies were diluted, if not stated differently, in 5% BSA and 0.1% NaN3 in TBS-T. Abbreviations: rb = rabbit, ms = mouse. * The phosphorylation sites in the human DNM1L are at positions Ser616 and Ser637. Due to a 100% sequence homology in the BLAST comparison of human with rat DNM1L, we assume that the antibody used also detect the rat-specific phosphorylation sites at Ser635 (Ser616, human) or Ser656 (Ser637, human). Since rat tissue was used in this manuscript, we termed the DNM1L phosphorylation sites according to the rat-specific positions.

    Journal: International Journal of Molecular Sciences

    Article Title: Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma

    doi: 10.3390/ijms232113618

    Figure Lengend Snippet: List of antibodies used for western blot. The antibodies were diluted, if not stated differently, in 5% BSA and 0.1% NaN3 in TBS-T. Abbreviations: rb = rabbit, ms = mouse. * The phosphorylation sites in the human DNM1L are at positions Ser616 and Ser637. Due to a 100% sequence homology in the BLAST comparison of human with rat DNM1L, we assume that the antibody used also detect the rat-specific phosphorylation sites at Ser635 (Ser616, human) or Ser656 (Ser637, human). Since rat tissue was used in this manuscript, we termed the DNM1L phosphorylation sites according to the rat-specific positions.

    Article Snippet: DNM1L (DRP1), phospho Ser637 * , rb , polyclonal , Origene , AP55855PU-S , 1:1000.

    Techniques: Western Blot, Sequencing