Journal: bioRxiv

Article Title: N-terminal phosphorylation inhibits Arabidopsis katanin and affects vegetative and reproductive development in opposite ways

doi: 10.64898/2026.01.20.700631

Figure Lengend Snippet: DDD has lower microtubule binding affinity and ATPase activity in vitro . (A) Representative Coomassie Blue-stained SDS-PAGE gels of microtubule co-sedimentation assays. 1 µM KTN1 (n = 3), DDD (n = 3), or AAA (n = 6) was incubated with the indicated concentration of taxol-stabilized microtubules. S, supernatant; P, pellet. Arrows identify the different protein bands. (B) Binding curves of KTN1, DDD, and AAA proteins corresponding to (A) . Each data point represents the mean ± SEM. Data were fit to a one-site saturation binding model to obtain the microtubule binding affinity and maximum amount of protein binding. (C-E) Plots of ATPase activity over time of 25 nM of KTN1 (C) , DDD (D) , and AAA (E) proteins. ATPase activity was measured as the amount of Pi release in the absence (black) or presence of either 25 nM (blue) or 250 nM microtubules (red). Each data point represents the mean ± SEM from 9 independent experiments.

Article Snippet: The ATPase activity of 25 nM of KTN1, DDD, or AAA in the presence of either 25nM or 250nM of taxol-stabilized microtubules was evaluated using a commercially available ATPase ELIPA Kit (Cytoskeleton #BK051) according to the manufacturer’s instructions.

Techniques: Binding Assay, Activity Assay, In Vitro, Staining, SDS Page, Sedimentation, Incubation, Concentration Assay, Protein Binding

Journal: Journal of Advanced Research

Article Title: Teleost GSDMEc regulates GSDMEa-mediated pyroptosis

doi: 10.1016/j.jare.2025.04.007

Figure Lengend Snippet: The interactions between NT216 and NT123/NT108 and their effects on NT-mediated pyroptosis. (A-C) HEK293T cells expressing mCherry-tagged NT261 and Flag-tagged NT123/NT108 alone or in various combinations were stained with Sytox Green and examined with a microscope (A), immunoblotted with the indicated antibodies (B), and measured for LDH release (C). Scale bar, 20 μm. Red arrows indicate pyroptotic cells. Values are shown as means ± SD. n = 3. *** P < 0.001. (D) HEK293T cells expressing NT261 and NT123/NT108 as above were observed with a microscope. Scale bar, 20 μm. (E and F) HEK293T cells expressing NT261 and NT123/NT108 as above were subjected to native- or SDS-PAGE and immunoblotting (IB) with the indicated antibodies (E). The cells were also subjected to immunoprecipitation (IP) using anti-Flag affinity gels and IB with the indicated antibodies (F). (G and H) HEK293T cells expressing mCherry-tagged NT261 and GFP-tagged NT123 (G) or CFP-tagged NT108 (H) were examined with a microscope. White arrows indicate protein aggregation. Scale bar, 5 μm. (I and J) HEK293T cells were transfected with empty vector (EV) or expressing Flag-tagged NT123 (I) or NT108 (J) for 24 h. Proteins from the whole cell, cytoplasm (Cyto), and membrane (Mem) were immunoblotted with antibodies against Flag, Na + /K + ATPase, or β-actin. (K and L) HEK293T cells expressing NT261 in various combinations with NT123 (K) or NT108 (L) for 15 h were extracted for cytoplasm/membrane proteins and immunoblotted as above. (M) HEK293T cells expressing NT261 in various combinations with NT123/NT108 for 24 h were subjected to immunoblot as above. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Polyclonal anti-Na + /K + ATPase antibody (14418-1-AP) was purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Staining, Microscopy, SDS Page, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Membrane

Journal: Journal of Advanced Research

Article Title: Teleost GSDMEc regulates GSDMEa-mediated pyroptosis

doi: 10.1016/j.jare.2025.04.007

Figure Lengend Snippet: The importance of the β2 strand to the function of NT261, NT123, and NT108. (A) Sequence alignment of NT123 and NT108. The predicted secondary structures of NT123 are indicated. Conserved residues are boxed in red. The residues selected for mutation are indicated with blue stars. (B and C) HEK293T cells expressing Flag-tagged NT261 in various combinations with Myc-tagged NT123/NT123M (B) or NT108/NT108M (C) were subjected to immunoprecipitation (IP) with anti-Flag affinity gels and immunoblotting (IB) with the indicated antibodies. (D) Proteins from the whole cell, cytoplasm (Cyto), and membrane (Mem) of HEK293T cells expressing Flag-tagged NT123 or NT123M were immunoblotted with antibodies against Flag, Na+/K + ATPase, or β-actin. (E) HEK293T cells expressing Flag-tagged NT261/NT261M together with Myc-tagged NT123 or NT108 were subjected to IP and IB as above. (F) HEK293T cells expressing Myc-tagged NT261 together with or without Flag-tagged NT261 or NT261M were subjected to IP and IB with antibodies against the indicated molecules. (G) HEK293T cells transfected with empty vector (EV) or expressing mCherry-tagged NT261 or NT261M were immunoblotted with anti-mCherry antibody following native- or SDS-PAGE. (H) HEK293T cells expressing Flag-tagged NT261 or NT261M were extracted for cytoplasm (Cyto)/membrane (Mem) proteins and immunoblotted with antibodies against Flag, Na+/K + ATPase, or β-actin. (I and J) HEK293T cells transfected with EV or expressing mCherry-tagged NT261 or NT261M were observed with a microscope after Sytox Green staining (I) and measured for LDH release (J). Values are shown as means ± SD. n = 3. *** P < 0.001. Scale bar, 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Polyclonal anti-Na + /K + ATPase antibody (14418-1-AP) was purchased from Proteintech (Wuhan, China).

Techniques: Sequencing, Mutagenesis, Expressing, Immunoprecipitation, Western Blot, Membrane, Transfection, Plasmid Preparation, SDS Page, Microscopy, Staining