Journal: iScience

Article Title: Rab27a promotes degradation of West Nile virus E protein in the lysosome

doi: 10.1016/j.isci.2024.109539

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Article Snippet: The cells were stained with rabbit anti-Rab27a antibody labeled with FlexAble CoraLite Plus 555 antibody (Proteintech) and rabbit ani-LAMP1 antibody labeled with FlexAble CoraLite Plus 405 antibody (Proteintech) for overnight at 4°C.

Techniques: Virus, Recombinant, Protease Inhibitor, Clone Assay, Luciferase, Software

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Interactome Analysis Identifies the Role of BZW2 in Promoting Endoplasmic Reticulum-Mitochondria Contact and Mitochondrial Metabolism

doi: 10.1016/j.mcpro.2023.100709

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Article Snippet: Samples were then incubated with imaging buffer containing primary Calnexin (CST, Rabbit) antibody (1:100) conjugated with FlexAble CoraLite Plus 405 (Proteintech), primary BZW2 (Abcam, Rabbit) antibody (1:100) conjugated with FlexAble CoraLite Plus 488, and primary VDAC (CST, rabbit) antibody (1:200) conjugated with FlexAble CoraLite Plus 647 (Proteintech) and then left to incubate for 2 h at room temperature.

Techniques: Clone Assay, Transfection, Transduction, Construct, Protease Inhibitor, Software, Calcium Assay, Isolation, Viability Assay, Sequencing, Modification, Western Blot, Bicinchoninic Acid Protein Assay

Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: ( a, b ) DOCK7 (TMW = 239 kDa, a ) and DNMT1 (TMW = 183 kDa, b ) protein interaction validated by DNMT1 co-immunoprecipitation and Western blot analysis by using a specific antibody against DOCK7 and DNMT1 in E14.5 cortical lysates. N = 3 experiments. + = DNMT1-antibody pulldown, - = IgG-antibody pulldown. ( c ) Representative images of N2a cells (grown for 48 h) co-stained with conjugated antibodies directed against DNMT1 (red) and DOCK7 (green), additionally stained with DAPI (blue), and captured by high-resolution STED microscopy. Scale bars: 5 µm. The white squares depict the magnification of the merge. Scale bars: 2 µm. Overlapping volumes are highlighted by white arrowheads. ( d, e ) Analysis of cytosolic colocalization between DNMT1 and DOCK7 by Pearson’s correlation coefficient (PCC) ( d ) and the percentage of colocalized volumes ( e ) in comparison to the corresponding rotated and randomized controls. n (ROIs) = 133; N (cells) = 45. ( f ) Predicted interaction between DNMT1 and DOCK7 using MD simulations and docking methods (left figure). Specific hot spot residues for the interaction between DNMT1 and DOCK7 are represented in the right figure. Hydrogen bonding and salt bridge interactions are shown as black dashes. ( g, h ) Inverted microphotographs of exemplary βIII-tubulin immunocytochemically stained cortical neurons (E14.5 + 2 DIV) transfected with control ( g ) or Dock7 ( h ) siRNA at 1 DIV for 24 h. Scale bars: 20 µm. ( i-l ) Analysis of morphological parameters, such as the length of the longest process ( i ), the number of processes ( j ), the branches per length summed across all processes likely representing dendrites ( k ), and the branches normalized to the longest process length likely representing axons ( l ). n (Ctrl siR) = 207 cells; n ( Dock7 siR) = 198 cells. N = 3 experiments. Two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Data are presented as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Immunoprecipitation, Western Blot, Staining, Microscopy, Comparison, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: (a) Representative STED micrographs of a fixed N2a cell cultured for 48 h, labeled with MitoTracker™ Deep Red FM (Mito., false color green), DAPI (blue), and an antibody against DOCK7 (red). Scale bar: 5 µm. The white box indicates the magnified region shown in the merge (Scale bar: 2 µm). Nuclear signals were computationally removed to improve visualization of the cytosolic compartment. (b) Quantification of cytosolic colocalization between DOCK7 and mitochondria by Pearson’s correlation coefficient (PCC) compared to a rotated randomized control. n (ROIs) = 45; N (cells) = 15. Two-tailed Student’s t -test, p < 0.0001. (c) Representative images of cortical neurons (P0 + 4 DIV) co-transfected at DIV 3 with Alexa Fluor™ 555–labeled control siRNA (red) and MT-GFP plasmid (green) and imaged 24 h post-transfection. Scale bar: 10 µm. (d) Exemplary tracking of MT-GFP–labeled mitochondria in cortical neurons following control, Dnmt1 , or Dock7 siRNA treatment. Non-motile mitochondria are marked by white arrowheads, motile mitochondria by colored stars (each color indicates one mitochondrion). White boxes in overview images (first panel) mark the magnified regions shown over time. The last frame (sixth panel) depicts temporally color-coded mitochondrial trajectories. Scale bars: 5 µm. (e) Representative kymographs illustrating mitochondrial motility under the respective conditions. (f) Quantification of the ratio of non-motile to motile mitochondria in cortical neurons (P0 + 4 DIV) following control, Dnmt1 , or Dock7 siRNA-mediated knockdown for 24 h at DIV 3. N (Ctrl siR) = 35 ROIs; n ( Dnmt1 siR) = 39 ROIs; n ( Dock7 siR) = 33 ROIs. N = 3 independent experiments. Kruskal–Wallis test followed by Dunn’s post hoc multiple comparison test, p < 0.01, p < 0.001. Data are shown as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Cell Culture, Labeling, Control, Two Tailed Test, Transfection, Plasmid Preparation, Knockdown, Comparison

Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: ( a, b ) Live cell imaging analysis capturing mitochondria accumulation using the MitoTracker TM Deep Red FM prior to branch formation in N2a cells 24 h after control, Dnmt1 , or Dock7 siRNA transfection. The white squares in the first panel indicate the magnified regions shown for each time frame. (a) Inverted grayscale images of mitochondria accumulation at branch initiation sites, Scale bars: 10 µm. The MitoTracker TM integrated density, normalized to the corresponding integrated density of the same position in the first frame, at prospective branchpoints is shown in ( b ). n (Ctrl siR) = 22 cells with 54 events; n ( Dnmt1 siR) = 13 cells with 24 events; n ( Dock7 siR) = 21 cells with 43 events. N = 4 experiments. Two-way ANOVA followed by Tukey’s post-hoc multiple comparison test, ** p < 0.01. ( c, d ) Analysis of the branch formation time ( c ) and exemplary tracking of a branching event (from the first mitochondria accumulation puncta to the formation of the branch) ( d ). The white squares in the first panel indicate the magnified regions shown for each time frame. Scale bars: 20 µm. n (Ctrl siR) = 22 cells with 54 events; n ( Dnmt1 siR) = 13 cells with 24 events; n ( Dock7 siR) = 21 cells with 43 events. N = 4 experiments. One-way ANOVA followed by Dunnett’s post-hoc multiple comparison test, ** p < 0.01, **** p < 0.0001. Data are presented as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Live Cell Imaging, Control, Transfection, Comparison

Journal: bioRxiv

Article Title: A cytosolic function of DNMT1 controls neuronal morphogenesis via microtubule regulation

doi: 10.1101/2025.10.19.683279

Figure Lengend Snippet: ( a, b ) Microphotographs of cortical neurons ( a, Scale bars: 20 µm) and N2a cells ( b, Scale bars: 40 µm), which were transfected with control, Dnmt1 , or Dock7 siRNA for 24 h after growing for one day, co-stained for DAPI (blue), ßIII-tubulin (TUBB3, green), and acetylated tubulin (AcTUB, magenta). ( c, d ) Analysis of the integrated density for the acetylated tubulin (AcTUB) normalized to the βIII-tubulin (TUBB3) integrated density after the respective knockdown in cortical neurons ( c ) and N2a cells ( d ). N = 3 experiments. One-way ANOVA followed by Dunnett’s post-hoc multiple comparison test, * p < 0.05, **** p < 0.0001. ( e, f ) Analysis of the acetylated tubulin (AcTUB) integrated density normalized to the βIII-tubulin (TUBB3) integrated density after the RG108 inhibitor treatment in cortical neurons ( e ) and N2a cells ( f ). N = 3 experiments. Two-tailed Student’s t-test, * p < 0.05. For ( c, e ): n (Ctrl siR) = 314 cells; n ( Dnmt1 siR) = 263 cells; n ( Dock7 siR) = 277 cells; n (DMSO) = 365 cells; n (RG108) = 358 cells. For ( d, f ): n (Ctrl siR) = 279 cells; n ( Dnmt1 siR) = 117 cells; n ( Dock7 siR) = 175 cells; n (DMSO) = 506 cells; n (RG108) = 525 cells. ( g, h ) Western blots revealing protein bands of STMN1 ( g , TMW = 17 kDa) and the phosphorylated version of STMN1 ( h , S16-P) (TMW = 17 kDa) in cortical single cell lysates (E14.5 + 2 DIV) treated previously with control, Dnmt1 , or Dock7 siRNA at 1 DIV for 24 h. γ-tubulin ( g , h , TUBG1, TMW = 51 kDa) was used as a housekeeper. ( i, j ) Analysis of the mean grey value of STMN1 normalized against TUBG1 ( i ) and the mean grey value of the phosphorylated version of STMN1 (S16-P) ( j ) normalized against STMN1. N = 3 experiments. One-way ANOVA followed by Dunnett’s post-hoc multiple comparison test, * p < 0.05, ** p < 0.01. Data are presented as mean ± SEM.

Article Snippet: For the colocalization studies between DNMT1 and DOCK7, antibodies were conjugated according to the FlexAble CoraLite® Plus 647 (Proteintech, USA, #KFA003) and FlexAble CoraLite® Plus 555 (Proteintech, USA, #KFA002) kits following the manufacturer’s protocol (2.5 μg antibody each).

Techniques: Transfection, Control, Staining, Knockdown, Comparison, Two Tailed Test, Western Blot