Journal: PLOS Biology

Article Title: Cdc42 interacts with chaperone Ydj1 to enhance its stability and partitioning during asymmetric cell division and aging in yeast

doi: 10.1371/journal.pbio.3003306

Figure Lengend Snippet: A. Cdc42-mCherry SW levels in each pair of young and old mothers and their daughters during M-cytokinesis ( n = 37 pairs, each group). **** p < 0.0001 from paired t-tests. Refer to legend. B. Cdc42-mCherry SW levels in mother and daughter pairs at the final division preceding death of both cells (see Cell 2 in ). n = 7 (out of 37 lineages); ns, p ≥ 0.05, paired t test. C. Schematic diagrams of GFP-Cdc42 and Cdc42-ritC-GFP with the Rit amphipathic helix (in blue). Levels of GFP-Cdc42 and Cdc42-ritC-GFP were compared by immunoblotting extracts from cells (expressing each allele as the sole genomic copy) grown at 25 °C to early log phase, using a monoclonal anti-GFP antibody and α-tubulin as a loading control. The mean GFP fluorescence in whole cells (mother and bud combined) of these strains, grown at 25 °C, is shown in a Tukey plot ( n = 72 per strain); ns, p ≥ 0.05, unpaired t t est. D. Localization of Cdc42-ritC-GFP at 25 °C, and quantification in mother and bud compartments during M-cytokinesis. Twenty representative pairs are shown. ns, p ≥ 0.05, paired t test. Scale bar: 3 µm. See and . The data underlying the graphs can be found in .

Article Snippet: Proteins were separated by SDS-PAGE on 12.5% polyacrylamide gels, and western blots were visualized using the LI-COR Odyssey system (LI-COR Biosciences, Lincoln, Nebraska) with the following antibodies: mouse monoclonal anti-Cdc42 antibody (clone 28-10) (EMD Millipore, MABN2485), rabbit polyclonal anti-RFP antibodies (Rockland, 600-401-379), mouse monoclonal anti-GFP antibodies (GFP-ID2-s) (DSHB, University of Iowa), and mouse monoclonal anti-alpha tubulin antibody (clone 12G10) (DSHB, University of Iowa).

Techniques: Western Blot, Expressing, Control, Fluorescence

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: Illustration of MACSima™ Imaging Cyclic Staining (MICS) principle MICS technology was applied (Step 46). (0) Image acquisition of 3D-IF staining in autofluorescence channel, followed by Photobleaching. (2–4) Multi-cyclic imaging: Rounds of 2D-IF staining with FITC, PE and APC coupled antibody fluorochrome-conjugate, image acquisition of respective FITC, PE and APC-channels and signal erasure by photobleaching.

Article Snippet: Timing: 7 days This step describes 3D-IF staining of target cells by passive diffusion at elevated temperatures and defined antibody-conjugate concentrations to improve homogeneous staining within large tissue samples., Note: Following steps have been optimized for Alexa Fluor 647 labeled anti-GFP nanobodies (see ) or 3D-IF antibodies provided by Miltenyi Biotec.

Techniques: Imaging, Staining

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: Timing: 7 days This step describes 3D-IF staining of target cells by passive diffusion at elevated temperatures and defined antibody-conjugate concentrations to improve homogeneous staining within large tissue samples., Note: Following steps have been optimized for Alexa Fluor 647 labeled anti-GFP nanobodies (see ) or 3D-IF antibodies provided by Miltenyi Biotec.

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Human OvCa) (A) Imaris 3D surface rendering of autofluorescence (cyan) and CD326 positive cells (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) with target plane in yellow. (C) Light sheet guided target plane selection representing CD326 positive cell (purple), CD45 positive cells (red), and CD3 positive cells (green). (D) DAPI overview image of selected tissue slice for 2D MACSima™ imaging. (E) Magnified merged six color multiparameter MICS image with CD45 (green), CD326 (cyan), FOLR1 (purple), Collagen III (red), Collagen IV (red), and CD31 (yellow). (F–L) Single staining MICS images (white) of DAPI (F), CD45 (G), CD326 (H), FOLR1 (I), Collagen III (J), Collagen IV (K), and CD31 (L) (gray) (see “Antibodies”). Scale bars: (A–F) 1 mm; (E) 250 μm; (F–L) 500 μm.

Article Snippet: Timing: 7 days This step describes 3D-IF staining of target cells by passive diffusion at elevated temperatures and defined antibody-conjugate concentrations to improve homogeneous staining within large tissue samples., Note: Following steps have been optimized for Alexa Fluor 647 labeled anti-GFP nanobodies (see ) or 3D-IF antibodies provided by Miltenyi Biotec.

Techniques: Imaging, Comparison, Selection, Staining

Journal: bioRxiv

Article Title: TurboID-based proteomic profiling reveals proxitome of the IRT1 metal transporter and new insight into metal uptake regulation in plants

doi: 10.64898/2026.03.16.712057

Figure Lengend Snippet: (A) Alphafold-based model prediction of the IRT1-TurboID fusion protein. IRT1, grey (with putative signal peptide in blue) ; TurboID, purple ; Flag tag, green. The TurboID insertion site (between S73 and R74) is shown in red. (B) Phenotype of 4-week-old wild-type (WT, Col), irt1 crispr mutant, and complemented irt1 /IRT1::IRT1-TurboID plants. (C) Immunoprecipitation of Lti6b-YFP. Immunoprecipitation was performed using anti-GFP antibodies on solubilized root protein extracts from 35S::Lti6b-YFP plants and subjected to immunoblotting with anti-GFP (left) or anti-IRT1 antibodies (right). Plants expressing Lti6b-YFP were grown for 14 days on iron-deficient conditions prior to protein extraction. Inputs and immunoprecipitated fractions are shown. IB, immunoblotting; IP, immunoprecipitation. The stain free signal is used as loading control. (D) Characterization of IRT1-TurboID and TurboID-Lti6b lines. Western blot analyses monitoring IRT1, IRT1-TurboID/TurboID-Lti6b and biotinylated protein accumulation in wild-type (WT), irt1 , irt1 /IRT1::IRT1-TurboID and IRT1::TurboID-Lti6b using anti-IRT1 antibodies, anti-FLAG antibodies, or avidin probe, respectively. Total proteins were extracted from roots of 14-day-old plants grown in half-strength MS media, treated with 100 µM Ferrozine for 24 hours, and subjected to with mock or 200 µM biotin in liquid medium for 4 hours. The stain free signal is used as loading control. (E) Influence of biotin addition of IRT1-TurboID ubiquitination profile. Immunoprecipitation of IRT1-TurboID was performed on root protein extracts probed using anti-ubiquitin antibodies (P4D1). Proteins were extracted from 14-day-old plants expressing IRT1::IRT1-TurboID treated with non-iron metals for 2 hours in liquid medium prior to addition of 200 µM biotin in non-iron metal liquid medium. (F) Alt Text : Illustration and data on the generation and characterization of IRT1 transgenic lines for TurboID.

Article Snippet: For protein detection, the following antibodies/probes were used: Monoclonal anti-GFP horseradish peroxidase-coupled (Miltenyi Biotech 130-091-833, 1/5,000), anti-ubiquitin P4D1 (Millipore 05-944, 1/2,500), anti-IRT1 (Agrisera AS11 1780, 1/5000), anti-FLAG M2 (Sigma-Aldrich, F1804, 1/2500), anti-RFP (Abcam AB34767, 1/5000), and avidin probe coupled to horse radish peroxidase (Sigma-Aldrich A3151, 1/5000).

Techniques: FLAG-tag, CRISPR, Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Protein Extraction, Staining, Control, Avidin-Biotin Assay, Ubiquitin Proteomics, Transgenic Assay

Journal: bioRxiv

Article Title: TurboID-based proteomic profiling reveals proxitome of the IRT1 metal transporter and new insight into metal uptake regulation in plants

doi: 10.64898/2026.03.16.712057

Figure Lengend Snippet: (A) Trimolecular fluorescence complementation (TriFC) assay in Nicotiana benthamiana leaves coexpressing ALFA NB-mCitN with AHA2-mCitC (top, positive control), NHX5-mCitC (middle) or RGLG2-mCitC (bottom) in presence (left) or absence (Ø, right) of IRT1-ALFA. The absence of IRT1-ALFA (Ø) serves as negative control. The fluorescence emanating from the constitutively-expressed and cotransformed MyrPalm-mCherry plasma membrane marker serves as internal control for ratiometric quantification in (B). Scale bars, 10 μm. (B) Ratiometric quantification of the mCitrine/MyrPalm-mCherry fluorescence signal ratios from confocal microscopy images of N. benthamiana shown in (A). Box-and-whisker plots indicate the median (line), interquartile range (box), 1.5 interquartile range (whiskers), and mean values (‘+’ symbol). Experiments were done in triplicates where six cells from two independent leaves were imaged. Data were analyzed by two-way ANOVA followed by Bonferroni multiple comparison post-test. Statistically significant differences between combinations (presence vs absence of IRT1-ALFA) are indicated by letters (P<0.05). Tested interactions were statistically different from negative controls. No statistical difference (n.s.) was observed among all tested interactors. (C) Interaction of IRT1 with NHX5 by coimmunoprecipitation using transient expression in N. benthamiana . Protein extracts from leaves expressing IRT1-mCit together with NHX5-mChe were subjected to immunoprecipitation with anti-GFP antibodies. The plasma membrane-localized MyrPalm-mChe reporter was used as negative control. Coimmunoprecipitation of NHX5-mChe was detected using anti-RFP antibodies. The stain free signal is used as loading control for inputs. (D) Interaction of IRT1 with RGLG2 by coimmunoprecipitation using transient expression in N. benthamiana . Protein extracts from leaves expressing IRT1-mCit or IRT1 2KR -mCit together with RGLG2-mChe or RGLG2 2CS -mChe were subjected to immunoprecipitation with anti-GFP antibodies. Lti6b-YFP was used as negative control. Coimmunoprecipitation of RGLG2-mChe or RGLG2 2CS -mChe was detected using anti-RFP antibodies. The stain free signal is used as loading control for inputs. (E) Phenotype of wild-type (WT), nhx5nhx6 , nhx5nhx6 /NHX5::NHX5-mChe, and nhx5nhx6 /UBI10::NHX5-mChe. (F) Tissue localization of NHX5 promoter activity. Confocal microscopy image of complemented plants expressing nhx5nhx6 /NHX5::NHX5-mChe. (G) Tissue localization of RGLG2 promoter. Confocal microscopy image of plants expressing GFP under the control of the RGLG2 promoter. (H) Subcellular localization and colocalization of IRT1-mCit and NHX5-mChe. Confocal microscopy images of roots from stable transgenic plants coexpressing PIN2::IRT1mCit and NHX5::NHX5-mChe. The M1 (IRT1 overlapping with NHX5) and M2 Manders’ (NHX5 overlap with IRT1) colocalization coefficients are shown (I) Subcellular localization and colocalization of transiently-expressed RGLG2-mChe in N. benthamiana with IRT1-mCit. The M1 (IRT1 overlapping with RGLG2) and M2 Manders’ (RGLG2 overlap with IRT1) colocalization coefficients are shown. (J) Alt Text : Microscopy images and blots validating the interaction between two candidate proximal proteins and IRT1.

Article Snippet: For protein detection, the following antibodies/probes were used: Monoclonal anti-GFP horseradish peroxidase-coupled (Miltenyi Biotech 130-091-833, 1/5,000), anti-ubiquitin P4D1 (Millipore 05-944, 1/2,500), anti-IRT1 (Agrisera AS11 1780, 1/5000), anti-FLAG M2 (Sigma-Aldrich, F1804, 1/2500), anti-RFP (Abcam AB34767, 1/5000), and avidin probe coupled to horse radish peroxidase (Sigma-Aldrich A3151, 1/5000).

Techniques: Fluorescence, Positive Control, Negative Control, Clinical Proteomics, Membrane, Marker, Control, Confocal Microscopy, Whisker Assay, Comparison, Expressing, Immunoprecipitation, Staining, Activity Assay, Transgenic Assay, Microscopy

Journal: bioRxiv

Article Title: TurboID-based proteomic profiling reveals proxitome of the IRT1 metal transporter and new insight into metal uptake regulation in plants

doi: 10.64898/2026.03.16.712057

Figure Lengend Snippet: (A) Root length phenotyping analysis of rglg1rglg2 in different non-iron metal conditions. Wild-type (WT) and rglg1rglg2 mutant plants were grown for 7 days on +/+ before being transferred for 7 days on the different metal conditions. Black lines shows the root length at the time of transfer. Scale bar, 1 cm. (B) Quantification of root elongation of wild-type (WT) and rglg1rglg2 mutant plants grown as in (A). Data are presented as ratio to the +/+ condition where IRT1 is not expressed. Experiments were done in triplicates (n=24). Error bars represent standard deviation. Asterisks indicate significant (two-way ANOVA , Sidakpost hoc test, ****P < 0.0001; **P < 0.01 ; ns, not significant). (C) Impact of the loss of RGLG1 and RGLG2 on IRT1 protein accumulation. Plants expressing UBI10::IRT1-mCit or rglg1rglg2 /UBI10-IRT1-mCit were treated with various non-iron metal provision and IRT1-mCit protein accumulation in root monitored by western blot using anti-GFP antibodies. The stain free signal is used as loading control. (D) Role of RGLG1 and RGLG2 on the non-iron metal-induced endocytosis or IRT1. Confocal microscopy images of plants coexpressing PIN2::IRT1-mCit and rglg1rglg2 /PIN2::IRT1-mCit treated various non-iron metal provision ( -/-, -/sub and -/+) for 3 h. Scale bar, 10 μm. (E) Quantification of the plasma membrane to intracellular fluorescence ratio of plants coexpressing PIN2::IRT1-mCit and rglg1rglg2 /PIN2::IRT1-mCit grown as in (D). Experiments were done in triplicates where five cells from three independent roots were imaged. Error bars represent standard deviation. Asterisks indicate significant (one-way ANOVA, Bonferroni’s multiple comparisons test, ****P <0.0001 ; ns, not significant). Alt Text : Microscopy data and phenotypic analyses characterizing the role of RGLG2 and RGLG1 as new IRT1 regulators.

Article Snippet: For protein detection, the following antibodies/probes were used: Monoclonal anti-GFP horseradish peroxidase-coupled (Miltenyi Biotech 130-091-833, 1/5,000), anti-ubiquitin P4D1 (Millipore 05-944, 1/2,500), anti-IRT1 (Agrisera AS11 1780, 1/5000), anti-FLAG M2 (Sigma-Aldrich, F1804, 1/2500), anti-RFP (Abcam AB34767, 1/5000), and avidin probe coupled to horse radish peroxidase (Sigma-Aldrich A3151, 1/5000).

Techniques: Mutagenesis, Standard Deviation, Expressing, Western Blot, Staining, Control, Confocal Microscopy, Clinical Proteomics, Membrane, Fluorescence, Microscopy

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 promoted the degradation and polyubiquitination of Snail. (A) UBR5 promoted the proteasomal degradation of Snail. HEK293T cells were transfected with Snail-Flag, Snail 6SA-Flag, UBR5-Myc, GFP, or empty vector and treated with DMSO, chloroquine, MG132, or CT99021 as indicated. The expression of Snail and GFP was assessed by western blotting. (B) UBR5 degraded Snail protein in a concentration-dependent manner. HEK293T cells were transfected with Snail-Flag, GFP, or in combination with different concentrations of wild-type and truncated UBR5-Myc for 48 h. Cell lysates were immunoblotted with anti-Snail antibodies. (C) UBR5 promoted K48 polyubiquitinated chain generation of Snail protein. In cellular ubiquitination assays, UBR5-Myc were co-transfected with Snail-Flag plasmids or with HA-Ub-K63 and HA-Ub-K48 plasmids. Western blotting was performed on cell lysates immunoprecipitated with an anti-Flag antibody, followed by the detection of polyubiquitination levels using an anti-Ub antibody. (D) UBR5 accelerated the Snail protein turnover through the HECT domain. HEK293T cells were transfected with corresponding plasmids. Cells were treated with cycloheximide (CHX) and harvested at indicated time points for immunoblotting with anti-Snail or anti-GFP antibody. The graph shows the quantification of Snail protein levels (based on the band intensity from the gels) normalized to those of GFP over the time course. Snail protein expression at the 0 h time point of treatment with CHX was set as 100 %. Experiments were performed in triplicate, and a representative experiment is presented.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Concentration Assay, Ubiquitin Proteomics, Immunoprecipitation

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 C2768S mutation abrogated the interaction with Snail. (A) His pull-down assays showed the abolished interactions between Snail and the UBR5 C2768S. A schematic representation of the UBR5 wild-type and C2768S mutation. (B) Co-immunoprecipitation assay showed that the interaction between the Snail and the UBR5 C2768S mutation was eliminated. HEK293T cells were transfected with UBR5-Myc, UBR5 C2768S-Myc, and Snail-Flag as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail and anti-UBR5 antibodies. (C) UBR5 C2768S abolished the UBR5-mediated degradation of Snail. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodies. (D) UBR5 C2768S did not accelerate Snail protein turnover. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc and treated with cycloheximide (CHX) as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodi.

Article Snippet: The membranes were probed with primary antibodies, including Flag (Proteintech, Wuhan, China, 66008-4-Ig), Myc (Proteintech, 60003-2-Ig), UBR5 (Proteintech, 66937-1-Ig), Snail (Santa Cruz Biotechnology, Oregon, USA, 166476), phosphorylated Snail (Biodragon, BD-PP0568), Slug (Santa Cruz Biotechnology, 271977), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (BD Transduction Laboratories, Franklin Lakes, USA, 610920), GSK3β (Proteintech, 82061-1-RR), pGSK3β (Proteintech, 67558-1-Ig), green fluorescent protein (GFP; Proteintech, 66002-1-Ig), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Bioss, Woburn, USA, 0978M).

Techniques: Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot