complex cyclosome apc c inhibitor protame  (MedChemExpress)

Journal: The Plant Cell

Article Title: Ubiquitin-dependent proteolysis of KNL2 driven by APC/C CDC20 is critical for centromere integrity and mitotic fidelity

doi: 10.1093/plcell/koaf164

Figure Lengend Snippet: Direct interaction of KNL2 with APC/C proteins. A) BiFC analysis showing the interactions between αKNL2/αKNL2-N/αKNL2-C fused to VENn and APC10/CDC20.1 fused to VENc. Venus fluorescence in the nucleus was shown in white dotted boxes. Scale bars represent 50 µ m. The right panel displays an enlarged image of the corresponding BiFC signals in the nucleus, but may not always correspond to the exact nuclei shown in the overview panel. Scale bars represent 5 µ m. B) Y2H assay testing interactions between αKNL2 (bait) and APC10/CDC20.1 (prey). Zygotes expressing both prey and bait were selected on -LT medium (double dropout: YNB without leucine and tryptophan). Protein–protein interactions were assessed on -LTH medium (TDO: YNB without leucine, tryptophan, and histidine). The strength of the protein–protein interactions was evaluated using a drop dilution assay. AD, activation domain; BD, binding domain. C) Co-IP interactions between αKNL2 and CDC20.1/APC10. N. benthamiana leaves were infiltrated with constructs containing CDC20.1-HA and αKNL2-C-cMYC (Lanes 1, 2), APC10-HA and αKNL2-C-cMYC (Lanes 3, 4), as well as HA and αKNL2-C-cMYC (Lanes 5, 6). Total protein extracts were precipitated with HA magnetic beads, and the samples were analyzed before (input) and after (IP) immunoprecipitation by immunoblotting with HA and c-MYC antibodies. The triangle (▵) marks the MW of CDC20.1, APC10, and empty control while the red arrowhead (▴) indicates the MW of αKNL2-C. IB, immunoblot; IP, immunoprecipitation. D) AlphaFold 3 prediction between C. elegans KNL-2 (blue) and MAT-3 or FZY-1 (yellow). The left panels present the predictions of the complexes formed between the protein pairs. Red boxes highlight the areas of interaction. The middle panels display heat maps of the interactions, with arrows indicating the specific sites of interaction. The right panels provide a close-up view of the red boxes in the left panels, highlighting the precise locations where the interaction is predicted. Ce MAT-3 (weakly) is predicted to interact with one of the predicted APC/C-specific degron motifs (purple), whereas Ce FZY-1 (strongly) predicted to interact with Ce KNL-2.

Article Snippet: For chemical treatments, 26S proteasome inhibitors, MG115 (MedChemExpress) and bortezomib (Selleckchem), and translation inhibitor, CHX (Selleckchem), were dissolved in DMSO and used at a concentration of 100 μ M. An APC/C inhibitor, Apcin (MedChemExpress), was used in different concentrations such as 5, 10, 15, 25, and 50 μ M. For analysis of αKNL2 degradation by the 26S proteasome, a plasmid carrying αKNL2-EYFP was infiltrated into the N. benthamiana plants and treated with the indicated concentrations of CHX, MG115, bortezomib, Apcin, or DMSO (control).

Techniques: Fluorescence, Y2H Assay, Expressing, Protein-Protein interactions, Dilution Assay, Activation Assay, Binding Assay, Co-Immunoprecipitation Assay, Construct, Magnetic Beads, Immunoprecipitation, Western Blot, Control

Journal: The Plant Cell

Article Title: Ubiquitin-dependent proteolysis of KNL2 driven by APC/C CDC20 is critical for centromere integrity and mitotic fidelity

doi: 10.1093/plcell/koaf164

Figure Lengend Snippet: αKNL2 is a target of the APC/C complex in A. thaliana. A) Immunostaining of meristematic nuclei from APC10-RNAi lines and wild-type plants transformed with the αKNL2-EYFP construct using anti-GFP antibodies. Scale bars represent 5 µ m. B and C) Analysis of αKNL2 protein levels in APC10-RNAi and wild-type plants expressing αKNL2-EYFP. B) Western blot analysis of nuclear protein extracts from APC10-RNAi and wild-type plants expressing αKNL2-EYFP, using an anti-GFP antibody. Tubulin was used as a loading control. The triangle symbol (▵) indicates the MW of αKNL2. C) Quantification of αKNL2 protein levels from B) , with band intensities normalized to tubulin. Data are presented as mean values ± SEM ( n = 3). Significant differences between groups were assessed using Welch's t -test and are marked by * ( P < 0.5). D) Immunostaining of meristematic nuclei of Arabidopsis wild-type (1) and the APC10-RNAi lines (2) using anti-αKNL2 antibodies. Scale bars are 5 μ m. E) Relative intensity measurements of αKNL2 signals on nuclei from APC10-RNAi and wild type. Boxplots show the distribution of fluorescence intensities ( n = 35 per group). The centerline indicates the median, and box limits represent the upper and lower quartiles (Q1 and Q3); whiskers extend to 1.5× the interquartile range. Significant differences between groups were assessed using Welch's t -test and are indicated by * ( P < 0.5). F) The mitotic localization of αKNL2 in root meristem tissues of wild-type (1,2) and APC10-RNAi (3,4) using αKNL2 antibodies (magenta). αKNL2 localizes to centromeres during mitosis in APC10-RNAi . DAPI-stained chromosomes are shown in blue, and anti-α-tubulin immunosignals are shown in green. (1, 3) metaphase; (2, 4) anaphase. Scale bars are 5 μ m. G and H) Analysis of endogenous αKNL2 protein levels in wild type and APC10-RNAi . G) Western blot analysis of nuclear protein extracts from wild type, and APC10-RNAi lines, detected using αKNL2-specific antibody. Tubulin was used as a loading control. The white triangle (▵) indicates the MW of αKNL2, and the red star (★) denotes the absence of degradation products. H) Quantification of αKNL2 protein levels from G) , with band intensities normalized to tubulin. Data are shown as mean values ± SEM ( n = 3). Significant differences between groups were assessed using Welch's t -test and are indicated by * ( P < 0.5).

Article Snippet: For chemical treatments, 26S proteasome inhibitors, MG115 (MedChemExpress) and bortezomib (Selleckchem), and translation inhibitor, CHX (Selleckchem), were dissolved in DMSO and used at a concentration of 100 μ M. An APC/C inhibitor, Apcin (MedChemExpress), was used in different concentrations such as 5, 10, 15, 25, and 50 μ M. For analysis of αKNL2 degradation by the 26S proteasome, a plasmid carrying αKNL2-EYFP was infiltrated into the N. benthamiana plants and treated with the indicated concentrations of CHX, MG115, bortezomib, Apcin, or DMSO (control).

Techniques: Immunostaining, Transformation Assay, Construct, Expressing, Western Blot, Control, Fluorescence, Staining

Journal: The Plant Cell

Article Title: Ubiquitin-dependent proteolysis of KNL2 driven by APC/C CDC20 is critical for centromere integrity and mitotic fidelity

doi: 10.1093/plcell/koaf164

Figure Lengend Snippet: The APC/C D-box1 region at the N-terminus is a functional degron of αKNL2. A) Schematic of the ubiquitination process involving E2 conjugation enzymes and E3 ligases, marking substrates with degrons for proteasomal degradation. B) Diagram of αKNL2 structure showing SBC, D-box1, and D-box2 degrons. C) WebLogo alignment showing conserved αKNL2 sequences (48 to 88 amino acids) in Brassicales, highlighting conserved SBC (VFTSS) and D-box1 (RGFL) domains (red asterisks). D) Superresolution SIM image showing colocalization of αKNL2 ΔD-box1 -EYFP fluorescence signals (green) with CENH3 (magenta) in N. benthamiana leaves. Scale bars represent 5 µ m. E) The localization pattern of αKNL2 ΔD-box1 -EYFP in Arabidopsis root tips. Scale bars represent 10 µ m. F) BiFC analysis shows no interaction between αKNL2 ΔD-box1 -VENn and CDC20.1-VENc (no fluorescence), while αKNL2 ΔD-box2 -VENn and CDC20.1-VENc show nuclear fluorescence. Scale bars represent 5 µ m. G) Y2H assay shows interaction of αKNL2 ΔD-box2 but not αKNL2 ΔD-box1 with CDC20.1. Zygotes were selected on -LT medium (double dropout), and interactions were evaluated on -LTH medium (TDO) using a drop dilution assay. AD, activation domain; BD, binding domain. H) Western blot of proteins from plants expressing αKNL2 ΔD-box1 -EYFP, αKNL2 ΔD-box2 -EYFP, and αKNL2-EYFP treated with Apcin, detected with anti-GFP antibody. The triangle (▵) represents αKNL2 MW. I) Quantification of protein levels from H) , normalized to tubulin, shown as mean ± SEM ( n = 3). Significant differences between groups were assessed using Welch's t -test and are indicated by ** ( P < 0.05).

Article Snippet: For chemical treatments, 26S proteasome inhibitors, MG115 (MedChemExpress) and bortezomib (Selleckchem), and translation inhibitor, CHX (Selleckchem), were dissolved in DMSO and used at a concentration of 100 μ M. An APC/C inhibitor, Apcin (MedChemExpress), was used in different concentrations such as 5, 10, 15, 25, and 50 μ M. For analysis of αKNL2 degradation by the 26S proteasome, a plasmid carrying αKNL2-EYFP was infiltrated into the N. benthamiana plants and treated with the indicated concentrations of CHX, MG115, bortezomib, Apcin, or DMSO (control).

Techniques: Functional Assay, Ubiquitin Proteomics, Conjugation Assay, Fluorescence, Y2H Assay, Dilution Assay, Activation Assay, Binding Assay, Western Blot, Expressing

Journal: The Plant Cell

Article Title: Ubiquitin-dependent proteolysis of KNL2 driven by APC/C CDC20 is critical for centromere integrity and mitotic fidelity

doi: 10.1093/plcell/koaf164

Figure Lengend Snippet: Model of APC/C-mediated ubiquitination and degradation of αKNL2 in Arabidopsis. When αKNL2 accumulates excessively, the APC/C E3 ubiquitin ligase, activated by E1 and E2 enzymes, targets it through the D-box1 motif (shown in red). This recognition triggers polyubiquitination at lysine residues 336 and 339, directing the protein to proteasomal degradation (upper part). αKNL2 is shown in green, while white circles with question marks indicate unknown regulatory mechanisms. Ubiquitin molecules are depicted as dark pink circles labeled “Ub,” and the APC/C–CDC20 complex is shown in light red-pink. During early mitosis, αKNL2 is also degraded through the APC/C–CDC20 pathway (dashed inhibitory arrows), ensuring proper SAC function and promoting cell cycle progression (lower part). This model was created using BioRender.com .

Article Snippet: For chemical treatments, 26S proteasome inhibitors, MG115 (MedChemExpress) and bortezomib (Selleckchem), and translation inhibitor, CHX (Selleckchem), were dissolved in DMSO and used at a concentration of 100 μ M. An APC/C inhibitor, Apcin (MedChemExpress), was used in different concentrations such as 5, 10, 15, 25, and 50 μ M. For analysis of αKNL2 degradation by the 26S proteasome, a plasmid carrying αKNL2-EYFP was infiltrated into the N. benthamiana plants and treated with the indicated concentrations of CHX, MG115, bortezomib, Apcin, or DMSO (control).

Techniques: Ubiquitin Proteomics, Labeling