Journal: bioRxiv

Article Title: Generation of a Synthetic Single Domain Antibody Library for Radiopharmaceutical Ligand Discovery

doi: 10.1101/2025.05.14.654066

Figure Lengend Snippet: A) Representative ELISA for Cal3 binding to CALR (Black circles), HER2 (pink squares), and 1% non-fat dry milk (teal triangles). Each point represents the mean of four individual measurements. Error bars denote standard deviation. B) Biolayer interferometry analysis of Cal3 binding to human CALR (hCALR). Individual curves representing different ligand binding concentrations from 23.8-22700 nM are depicted.

Article Snippet: To generate a capture antigen, 50 µg lyophilized recombinant human calreticulin (CALR) (Sino Biological) was reconstituted to 0.2 mg/mL in PBS and incubated with 10 molar excess of 10mM EZ-Link NHC-LC-Biotin (ThermoFisher) at 37C with shaking at 250 rpm for 1 hour.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Standard Deviation, Ligand Binding Assay

Journal: bioRxiv

Article Title: Generation of a Synthetic Single Domain Antibody Library for Radiopharmaceutical Ligand Discovery

doi: 10.1101/2025.05.14.654066

Figure Lengend Snippet: A) Representative PET/CT images of 64 Cu-NOTA-Cal3 1-hour post-injection via tail vein. Male C57/Bl6 (n=4) are bearing hCALR- or BSA-Matrigel plugs on the left and right shoulders, respectively. D) Quantification of PET signal from CALR and BSA plugs. C) Quantification of PET signal from clearance-related organs.

Article Snippet: To generate a capture antigen, 50 µg lyophilized recombinant human calreticulin (CALR) (Sino Biological) was reconstituted to 0.2 mg/mL in PBS and incubated with 10 molar excess of 10mM EZ-Link NHC-LC-Biotin (ThermoFisher) at 37C with shaking at 250 rpm for 1 hour.

Techniques: Positron Emission Tomography-Computed Tomography, Injection

Journal: bioRxiv

Article Title: The DBL-1/TGF-β signaling pathway regulates pathogen-specific innate immune responses in C. elegans

doi: 10.1101/2021.03.30.437693

Figure Lengend Snippet: Loss of DBL-1 decreases lifespan of animals exposed to Gram-negative and Gram-positive bacteria. Wild-type and dbl-1(−) animals were scored for survival over time from the L4 stage (t=0 hours) on the following bacteria: a) E. coli OP50 (control), E. cloacae , c) K. oxytoca , d) S. marcescens , e) B. megaterium , f) E. faecalis , and g) S. epidermidis . Survival fraction was calculated by the Kaplan-Meier method. p -values were calculated using log-rank test and p <0.01 compared to wild-type animals exposed to the same bacteria was considered significant. One representative trial of at least three is presented.

Article Snippet: The bacterial strains used in this study include Bacillus megaterium (Carolina Biological Supply Company), Escherichia coli (OP50), Enterobacter cloacae (49141TM), Enterococcus faecalis (51299TM), Klebsiella oxytoca (49131TM), Serratia marcescens (Carolina Biological Supply Company), and Staphylococcus epidermidis (49134TM).

Techniques: Bacteria, Control

Journal: bioRxiv

Article Title: The DBL-1/TGF-β signaling pathway regulates pathogen-specific innate immune responses in C. elegans

doi: 10.1101/2021.03.30.437693

Figure Lengend Snippet: Loss of DBL-1 and exposure to specific bacteria results in decreased pharyngeal pumping. Wild-type and dbl-1(−) animals were exposed to the following bacteria: a, b) E. coli OP50 (control); a) E. cloacae , K. oxytoca , S. marcescens ; b) B. megaterium , E. faecalis , or S. epidermidis at the L4 stage. After 48 hours of exposure, the number of pharyngeal pumps were counted twice per 20 seconds. The pharyngeal pumps were averaged for each animal. One representative trial of at least three is presented. Error bars represent standard deviation. n=12 per condition. * p <0.01, ns not significant, compared to wild-type animals exposed to the same bacteria, and # p <0.01, respective genotype exposed to test bacteria in comparison to control bacteria by two-way ANOVA followed by unpaired t -test.

Article Snippet: The bacterial strains used in this study include Bacillus megaterium (Carolina Biological Supply Company), Escherichia coli (OP50), Enterobacter cloacae (49141TM), Enterococcus faecalis (51299TM), Klebsiella oxytoca (49131TM), Serratia marcescens (Carolina Biological Supply Company), and Staphylococcus epidermidis (49134TM).

Techniques: Bacteria, Control, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: The DBL-1/TGF-β signaling pathway regulates pathogen-specific innate immune responses in C. elegans

doi: 10.1101/2021.03.30.437693

Figure Lengend Snippet: Avoidance to Gram-negative bacteria increases upon loss of DBL-1 signaling. Wild-type and dbl-1(−) animals at the L4 stage (t=0 hours) were exposed to the following bacteria: a) E. coli OP50 (control), b) E. cloacae , c) K. oxytoca , d) S. marcescens , e) B. megaterium , f) E. faecalis , or g) S. epidermidis . Avoidance of animals to the bacteria was monitored over time. The avoidance ratio was calculated and compared between wild-type and dbl-1(−) animals. One representative trial of three is presented. Error bars represent standard deviation. n=30 per condition per trial. * p <0.05, and ns not significant, compared to wild-type animals exposed to the same bacteria by repeated measures ANOVA using Tukey’s post-hoc test.

Article Snippet: The bacterial strains used in this study include Bacillus megaterium (Carolina Biological Supply Company), Escherichia coli (OP50), Enterobacter cloacae (49141TM), Enterococcus faecalis (51299TM), Klebsiella oxytoca (49131TM), Serratia marcescens (Carolina Biological Supply Company), and Staphylococcus epidermidis (49134TM).

Techniques: Bacteria, Control, Standard Deviation

Journal: bioRxiv

Article Title: The DBL-1/TGF-β signaling pathway regulates pathogen-specific innate immune responses in C. elegans

doi: 10.1101/2021.03.30.437693

Figure Lengend Snippet: Avoidance to Gram-negative bacteria increases upon loss of canonical DBL-1 signaling. Wild-type, sma-2(−) , sma-3(−) , and sma-4(−) animals at the L4 stage (t=0 hours) were exposed to the following bacteria: a) E. coli OP50 (control), b) E. cloacae , b) K. oxytoca , d) S. marcescens , e) B. megaterium , f) E. faecalis , or g) S. epidermidis . Avoidance of animals to the bacteria was monitored over time. The avoidance ratio was calculated and compared between wild-type and Smad mutant animals. Avoidance ratio = number of animals off the bacterial lawn/ total number of animals. One representative trial of three is presented. Error bars represent standard deviation. n=30 per condition per trial. * p <0.05, ns not significant, compared to wild-type animals exposed to the same bacteria by repeated measures ANOVA using Tukey’s post-hoc test.

Article Snippet: The bacterial strains used in this study include Bacillus megaterium (Carolina Biological Supply Company), Escherichia coli (OP50), Enterobacter cloacae (49141TM), Enterococcus faecalis (51299TM), Klebsiella oxytoca (49131TM), Serratia marcescens (Carolina Biological Supply Company), and Staphylococcus epidermidis (49134TM).

Techniques: Bacteria, Control, Mutagenesis, Standard Deviation

Journal: bioRxiv

Article Title: The DBL-1/TGF-β signaling pathway regulates pathogen-specific innate immune responses in C. elegans

doi: 10.1101/2021.03.30.437693

Figure Lengend Snippet: Smad transcription factors gene expression is altered by specific bacteria. Wild-type and dbl-1(−) animals at the L4 stage were exposed to E. coli OP50 (control), E. cloacae , K. oxytoca , S. marcescens , B. megaterium , E. faecalis , or S. epidermidis for 48 hours. mRNA expression levels of a-d) sma-2 , e-h) sma-3 , and i-l) sma-4 were quantitated by real-time PCR. Experiments were performed in triplicate and in three independent trials. One representative trial of three is presented. Error bars represent standard deviation. * p <0.05 in a, b, e, f, i, j, mRNA expression level in dbl-1(−) population compared to wild-type population exposed to the same bacteria by unpaired t -test. * p <0.05 in c, d, g, h, k, l, mRNA expression level in respective genotype exposed to test bacteria compared to control bacteria by unpaired t -test.

Article Snippet: The bacterial strains used in this study include Bacillus megaterium (Carolina Biological Supply Company), Escherichia coli (OP50), Enterobacter cloacae (49141TM), Enterococcus faecalis (51299TM), Klebsiella oxytoca (49131TM), Serratia marcescens (Carolina Biological Supply Company), and Staphylococcus epidermidis (49134TM).

Techniques: Gene Expression, Bacteria, Control, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: bioRxiv

Article Title: The DBL-1/TGF-β signaling pathway regulates pathogen-specific innate immune responses in C. elegans

doi: 10.1101/2021.03.30.437693

Figure Lengend Snippet: DBL-1 signaling is activated upon exposure to Gram-negative bacteria but is repressed in response to Gram-positive bacteria. L4 animals expressing the RAD-SMAD reporter in a wild-type background were exposed to a, h) E. coli OP50 (control), b, h) E. cloacae , c, h) K. oxytoca , d, h) S. marcescens , e) B. megaterium , f) E. faecalis , or g) S. epidermidis and L2-stage progeny were imaged. Mean RAD-SMAD fluorescence intensity of five hypodermal nuclei per animal was quantitated and compared. Experiments were performed in three independent trials. One representative trial is presented. Error bars represent 95% confidence intervals. n=15 per condition in each trial. * p <0.05, mean fluorescence intensity in wild-type background on test bacteria compared to control bacteria by unpaired t -test. Scale bar, 10 μm.

Article Snippet: The bacterial strains used in this study include Bacillus megaterium (Carolina Biological Supply Company), Escherichia coli (OP50), Enterobacter cloacae (49141TM), Enterococcus faecalis (51299TM), Klebsiella oxytoca (49131TM), Serratia marcescens (Carolina Biological Supply Company), and Staphylococcus epidermidis (49134TM).

Techniques: Bacteria, Expressing, Control, Fluorescence

Journal: bioRxiv

Article Title: The DBL-1/TGF-β signaling pathway regulates pathogen-specific innate immune responses in C. elegans

doi: 10.1101/2021.03.30.437693

Figure Lengend Snippet: Expression of innate immunity genes is differentially regulated by DBL-1 signaling in different bacterial environments. L4 wild-type and dbl-1(−) animals were exposed for two days to E. coli OP50 (control), S. marcescens , or E. faecalis . RNA-seq analysis reveal significant changes (adjusted p -value <0.01) in gene expression of animals lacking DBL-1. Heatmaps show differential innate immunity gene expression in animals lacking DBL-1 exposed to a) E. coli OP50, b) S. marcescens , and c) E. faecalis in comparison to wild-type animals exposed to the same bacteria. Average log FPKM values from three independent trials are represented.

Article Snippet: The bacterial strains used in this study include Bacillus megaterium (Carolina Biological Supply Company), Escherichia coli (OP50), Enterobacter cloacae (49141TM), Enterococcus faecalis (51299TM), Klebsiella oxytoca (49131TM), Serratia marcescens (Carolina Biological Supply Company), and Staphylococcus epidermidis (49134TM).

Techniques: Expressing, Control, RNA Sequencing, Gene Expression, Comparison, Bacteria

Journal: bioRxiv

Article Title: The DBL-1/TGF-β signaling pathway regulates pathogen-specific innate immune responses in C. elegans

doi: 10.1101/2021.03.30.437693

Figure Lengend Snippet: Innate immune reporter activity is regulated by exposure to specific bacteria and by DBL-1 signaling. Comparison of a, b) dod-22:: GFP, c, d) F55G11.7 :: GFP, e, f) irg-4:: GFP, g, h) dod-24p:: GFP, and i, j) ilys-3p:: GFP intensities in adult wild-type and dbl-1(−) animals after a two-day exposure to the following bacteria; control E. coli OP50, E. cloacae , K. oxytoca, S. marcescens, B. megaterium , E. faecalis , or S. epidermidis . Imaging conditions including exposure times were consistent with respective control. Three independent trials were performed. One representative trial is shown. Error bars represent standard deviation. n= at least 15 per condition in each trial. * p <0.05 compared to wild-type animals exposed to the same bacteria and # p <0.05 respective genotype exposed to test bacteria in comparison to control bacteria, by two-way ANOVA followed by unpaired t -test.

Article Snippet: The bacterial strains used in this study include Bacillus megaterium (Carolina Biological Supply Company), Escherichia coli (OP50), Enterobacter cloacae (49141TM), Enterococcus faecalis (51299TM), Klebsiella oxytoca (49131TM), Serratia marcescens (Carolina Biological Supply Company), and Staphylococcus epidermidis (49134TM).

Techniques: Activity Assay, Bacteria, Comparison, Control, Imaging, Standard Deviation

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Schematic of the BioID study of WDR62-interacting proteins. ( B ) Validation of WDR62-BirA*-HA localisation in AD293 cells by fluorescence microscopy. ( C ) Streptavidin-coupled beads were used to capture bioinylated proteins, and subsequent immunoblot confirms MAPKBP1, AURKA, JNK1/2, CEP170 and BAG2 as interactors of WDR62 in asynchronous and mitotically arrested cells. ( D ) Venn diagram of published WDR62 interactors (BioGRID database), interactors identified through BioID screening, and interactors found in both datasets. ( E ) BioID-identified interactors were sorted into those identified in asynchronous or mitotic cells, or both. ( F – H ) Gene ontology (GO) analysis of significantly enriched ( F ) biological processes, ( G ) molecular functions, and ( H ) cellular components mediated/localised by WDR62 interactors. GO term enrichment was performed using the PANTHER over-representation test (Fisher’s exact test, FDR-corrected). The x -axis shows -log 10 (FDR), labelled as −log 10 (P). ( I ) STRING-based reconstruction of the network of proteins identified as interactors of WDR62 by our BioID study. Yellow lines indicate empirically confirmed interactions, grey lines are interactions curated in the STRING protein interaction database. Data represent n = 3 independent replicates. Scale bars represent 10 µm. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Biomarker Discovery, Fluorescence, Microscopy, Western Blot

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Top-ranked model predicted by AlphaFold2 between WDR62 and BAG2 with calculated mean predicted local distance difference test (pLDDT), predicted template modelling (pTM) and interphase predicted template modelling (ipTM) scores. ( B ) Predicted aligned error (PAE) plot of predicted WDR62-BAG2 heterodimer. X - and y - axes show indexed residues of corresponding subunits, as indicated. Aligned error in angstroms (Å) is colour coded (green = low PAE (high confidence), white = high PAE (low confidence). ( C ) Schematic depicting WDR62(FL) (aa 1–1523), truncated mutants WDR62(N) (1-841), WDR62(C) (842–1523), and microcephaly-associated mutants WDR62(3936dupC) (1–1329), V65M and R438H. ( D ) GFP-trap immunoprecipitation of WDR62(FL)-, WDR62(N)- and WDR62(C)-EGFP and immunoblot for GFP and endogenous BAG2 (* P = 0.0372). ( E ) GFP-trap immunoprecipitation of WDR62(FL)-, WDR62(R438H)-, WDR62(V65M), and WDR62(3936dupC)-EGFP and immunoblot for GFP and endogenous BAG2 (* P = 0.0108, ** P = 0.0089). Densitometric BAG2 band values were normalised and expressed as BAG2/GFP ratio for ( D , E ) represented underneath the blots. Data represent mean ± SEM of n > 3 independent replicates. P values calculated based on mean values using a one-way ANOVA (* P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s. is P > 0.05). .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Immunoprecipitation, Western Blot

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Representative images of AD293 cells expressing WDR62-mCherry, showing WDR62 de-mixes into cytoplasmic puncta following treatment with 0.5 M sorbitol, dextrose, or sucrose for 1 h. ( B ) Titration of WDR62 granule assembly. A concentrated solution of sorbitol was added dropwise to a live-cell culture dish containing AD293 cells expressing WDR62-mCherry. Sorbitol was added every minute for the duration of the experiment (10 min). Total sorbitol concentration in culture media is displayed in the figure. The blue line represents a quantification of the total WDR62 condensation area over time. Condensed area is the total sum of WDR62-mCherry expression, normalised by its highest value. Scale bar represents 5 µm. ( C ) Three-dimensional orthogonal projection of WDR62 granules. ( D ) Average circularity of WDR62 granules. A circularity of 1 is equivalent to a perfect circle. ( E ) The average proportion (%) of cells containing WDR62 granules before and after sorbitol stress. Two-tailed unpaired Student’s T test (**** P < 0.0001). ( F ) The average diameter (µm) and number per cell of WDR62 granules. ( G ) Live-cell imaging experiments reveal that WDR62 granules rapidly assemble (~1 min) and disassemble (~10 s) following the addition and removal of sorbitol stress. Bottom represents the kymograph corresponding to the white dashed line on the first image. ( H ) WDR62 granules undergo fission and fusion events. ( I ) The left graph shows the trajectory of both granules over the time-lapse experiment, the right graph shows the area of both granules before (0–100 s), during (100–125 s) and after merging (125–350 s). ( J ) AD293 cells transiently transfected with WDR62-mCherry and stress granule marker, EGFP-G3BP, pre-treated with 100 µg/ml cycloheximide (CHX) ( + ) or vehicle control (−) for 1 h. Cells were subsequently treated with 0.5 mM sodium arsenite or 0.5 M sorbitol. Representative confocal micrographs demonstrate minimal co-localisation between WDR62-mCherry and G3BP-EGFP, indicated by fluorescence intensity plots ( y -axis represents fluorescence intensity (a.u.), x -axis (µm) represents length of white line drawn on ROI image. ( K ) Bar graph representing the proportion (%) of cells containing WDR62 granules following treatments in ( J ). (**** P < 0.0001). ( L ) Bar graph representing the proportion (%) of cells containing G3BP1 granules following treatments in ( J ). (* P = 0.0115, *** P = 0.0007, **** P < 0.0001). Data represent mean ± SEM of n = 4 independent replicates. P values calculated based on mean values using a one-way ANOVA (* P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s. is P > 0.05). All scale bars represent 20 µm. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Expressing, Titration, Cell Culture, Concentration Assay, Two Tailed Test, Live Cell Imaging, Transfection, Marker, Control, Fluorescence

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: Representative confocal micrographs of AD293 cells treated with 0.5 M sorbitol for 1 h. Cells transiently transfected with WDR62-mCherry and either the HSP co-chaperones ( A ) BAG2-EGFP, ( B ) EGFP-DNAJC7, ( C ) EGFP-STIP1, or de novo purine biosynthesis (DNPB) enzymes ( D ) PFAS-EGFP, ( E ) PPAT-EGFP, ( F ) GART-EGFP or ( G ) PAICS-EGFP. Co-localisation of each signal is indicated by fluorescence intensity plots to the right of each set of images ( y -axis represents fluorescence intensity (a.u.), x -axis (µm) represents the length of the white line drawn on the ROI). ( H ) Bar graph representing the co-localisation between WDR62-mCherry and EGFP signal for each respective protein (mean ± SD). Each dot represents the Person’s correlation coefficient for a single ROI. ( I ) Confocal micrographs of AD293 cells co-transfected with WDR62-mCherry and PFAS-EGFP, in either control (top) or purine-depleted (bottom) conditions. Bar graph on the right represents co-localisation between WDR62-mCherry and PFAS-EGFP (mean ± SD) ( ***P = 0.0004). ( J ) Co-immunoprecipitation and immunoblot of myc-PFAS and HA-WDR62. ( K ) The association of endogenous WDR62 with BAG2 and PFAS as measured by PLA (grey spots) with DAPI-stained nuclei in blue. PLA signal is observed under basal (control) and sorbitol-treated (0.5 M, 1 h) conditions. ( L ) Quantification of the number of PLA puncta per cell for WDR62/BAG2 and WDR62/PFAS. Data represent n = 3 biological replicates. P values calculated based on mean values using a one-way ANOVA for ( H ) and a two-tailed unpaired T test for ( I , L ) (* P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s. is P > 0.05). All scale bars represent 20 µm. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

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

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Western blot confirming the loss of WDR62 expression in WDR62 KO cells. ( B ) XTT assay quantification of cell proliferation (A 490nm -A 690nm ) of WT and KO cells cultured for 0, 4 and 7 days in purine-rich or purine-depleted media. Day 4 ( **P = 0.0028), Day 7 (** P = 0.0024). ( C ) Representative images of WT and WDR62 KO cells cultured in purine-rich or purine-depleted media. Bar graphs depict the proportion (%) of rounded cells in each category. Day 4 ( **P = 0.0081), Day 7 (** P = 0.0013). ( D ) LDH assay of percentage cytotoxicity (% cytotoxicity) of WT and KO cells cultured for 7 days in purine-rich or purine-depleted media (** P = 0.0027, **** P < 0.0001). ( E ) qPCR of WDR62, de novo purine biosynthesis ( PRPS1, PPAT, GART, PFAS, PAICS, ADSL, ATIC ) purine salvage ( ITPA, ADA, HPRT1, IMPDH2, AK) and purine degradation ( XDH) gene expression in WT and WDR62 KO AD293 cells. PRPS1 (* P = 0.0133), P FAS **( P = 0.0066), IMPDH2 (* P = 0.049), H P RT1 (** P = 0.005), XDH (* P = 0.0109). ( F ) Schematic of incorporation of 13 C from 13 C-glycine into the purine ring. Bar graphs of normalised peak areas of 13 C[M + 2] labelled purine metabolites, as measured by LC-MS/MS in WT and KO AD293 cells when cultured in purine-rich conditions. 13 C-AMP[M + 2] (* P = 0.0296), 13 C-ADP[M + 2] (**** P < 0.0001), 13 C-ATP[M + 2] (*** P = 0.0008), 13 C-GDP[M + 2] (*** P = 0.0002), 13 C-GTP[M + 2] (** P = 0.0062). ( G ) Schematic of incorporation of 13 C from 13 C-hypoxanthine into the purine ring. Bar graphs of normalised peak areas of 13 C[M + 5] labelled purine metabolites, as measured by LC-MS/MS in WT and KO AD293 cells when cultured in purine-rich conditions. Asterisks denote statistical significance (two-tailed unpaired T test) in comparison of WT and KO groups for each target gene. Data represent n = 8 independent replicates for ( B – D ), n = 4 for ( E ), and n = 5 for ( F , G ). P values calculated based on mean values using a one-way ANOVA for ( B-D ) or two-tailed unpaired T tests for ( E – G ). (* P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s. is P > 0.05). All data represent mean ± SEM. Scale bars represent 100 µm. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Western Blot, Expressing, XTT Assay, Cell Culture, Lactate Dehydrogenase Assay, Gene Expression, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Comparison

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Immunoblot of purine metabolic enzymes PFAS and HPRT.and co-chaperone BAG2 in WT and WDR62 KO AD293 cells. BAG2 (** P = 0.0046), HPRT (*** P = 0.0003). ( B ) Immunoblot of WDR62 KO AD293 cells rescued with EGFP only, WDR62(FL)-EGFP, or mutants WDR62(R438H)-EGFP and WDR62(3936dupC)-EGFP (* P = 0.0464, ** P = 0.0026, *** P = 0.0002). ( C ) Cycloheximide chase assay examining the half-life of HPRT, PFAS and BAG2 in WT and WDR62 KO cells. WT and WDR62 KO cells treated with 100 µM CHX for 0, 24, or 48 h (* P = 0.0196). ( D , F ) Immunofluorescence and confocal micrographs of endogenous HPRT in WT and WDR62 KO cells treated with vehicle (DMSO), or the HSP90 inhibitors NVP-AUY922 or 17-AAG (500 nM, 1 h). Far right image represents pseudo-coloured “fire” LUT to better visualise pixel intensity. Fluorescence intensity plots to the right of each set of images ( y -axis represents fluorescence intensity (a.u.), x -axis (µm) represents the length of the white line drawn on pseudo-coloured intensity image. ( E , G ) SuperPlots of averaged normalised (per µm) standard deviation (SD) of fluorescence intensity (a.u.) along random lines plotted in the cytoplasm of random cells. Higher SD values indicate greater heterogeneity in fluorescence intensity, characteristic of a less diffuse and more punctate distribution. WT vs. KO untreated (*** P = 0.0004), WT vs. KO DMSO (*** P = 0.0002), KO DMSO vs. KO NVP (* P = 0.0151), KO DMSO vs. KO AAG (* P = 0.0157). ( H ) Immunofluorescence and confocal micrographs of endogenous HPRT with WDR62-mCherry in control and sorbitol-treated (0.5 mM, 1 h) cells ( **P = 0.001). ( I – K ) The association of endogenous WDR62 with HPRT, or endogenous HPRT with BAG2 as measured by PLA (grey spots) with DAPI-stained nuclei in blue. PLA signal is observed under control or sorbitol-treated conditions for ( I ) (* P = 0.0385), or control siRNA or BAG2 siRNA conditions for ( J ) (* P = 0.0147), and WT and WDR62 KO conditions for ( K ). Quantification of the number of PLA puncta per cell represented in SuperPlots on the right of the images. ( L ) Western blot analyses of WDR62, BAG2, HPRT and PFAS expression in WT and WDR62 KO cells treated with non-targeting or BAG2 siRNA. IB: BAG2, non-targeting siRNA, WT vs. KO (* P = 0.017), IB: BAG2, WT cells, non-targeting vs. BAG2 siRNA (* P = 0.0469), IB: BAG2, KO cells, non-targeting vs. BAG2 siRNA (*** P = 0.0002), IB: HPRT, non-targeting siRNA, WT vs. KO (*** P = 0.0006), IB: HPRT, KO cells, non-targeting vs. BAG2 siRNA (* P = 0.0331). Data represent n = 10 independent replicates for ( A ), n = 5 for ( B ), n = 9 for ( C ), n = 4 for ( D , E , K , L ), and n = 3 for ( F , G , H , I , J ). P values calculated based on mean values using a one-way ANOVA for ( B , C , G , H , L ) or a two-tailed unpaired T test for ( A , E , I , J , K ) (* P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, n.s. is P > 0.05). All scale bars represent 20 µm. All data represent mean ± SEM. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Western Blot, Immunofluorescence, Fluorescence, Standard Deviation, Control, Staining, Expressing, Two Tailed Test

Journal: The EMBO Journal

Article Title: Microcephaly-associated protein WDR62 supports purine metabolism by interacting with co-chaperone BAG2

doi: 10.1038/s44318-026-00724-0

Figure Lengend Snippet: ( A ) Immunoblots of (left to right) WDR62, HPRT, and BAG2 in Neuro2a cells transfected with no siRNA, non-targeting siRNA, or siRNA against Wdr62 , Hprt , or Bag2 , respectively. Bar graphs to the right of each immunoblot represent densitometric quantification of signal over n = 3 biological replicates (* P = 0.0199, ** P = 0.0025). ( B – D ) In utero electroporation of control siRNA or siRNA targeting Wdr62 , Hprt , or Bag2 into E14.5 cortices, followed by analysis at E16.5. ( B ) Distribution of electroporated GFP-positive (GFP + ) cells in the ventricular zone/subventricular zone (VZ), intermediate zone (IZ), cortical plate (CP), or marginal zone (MZ). ( C ) Immunostaining electroporated brains sections for Ki67 and PH3 as markers of cell proliferation and mitosis respectively in electroporated brain sections. ( D ) Analysis of cell proliferation (Ki67 + /GFP + ) and ( E ) mitotic (PH3 + /GFP + ) cells in cortex, IZ and the VZ/SVZ, the latter delineated in half between the region closest to the apical surface of the lateral ventricles (lower) and away from the apical region (upper). (Ki67 + /GFP + )/GFP + , control vs. Wdr62 si (* P = 0.0275), (Ki67 + /GFP + )/GFP + , control vs. Hprt si (* P = 0.0281), (Ki67 + /GFP + )/GFP + in upper VZ/SVZ, control vs. Hprt si (** P = 0.0081), (Ki67 + /GFP + )/GFP + in IZ, control vs. Hprt si (* P = 0.0338). ( F ) Analysis of cell differentiation into intermediate progenitors (TBR2 + /GFP + ) and TBR2-negative progenitors (TBR2 - /GFP + ) in electroporated brain sections. (TBR2 + /GFP + )/GFP + in VZ/SVZ (* P = 0.0213), (TBR2 - /GFP + )/GFP + in VZ/SVZ (* P = 0.0236). All scale bars represent 50 µm. All data represent mean ± SEM. .

Article Snippet: WDR62 (rabbit polyclonal) , Bethyl Laboratories , A301-560A.

Techniques: Western Blot, Transfection, In Utero, Electroporation, Control, Immunostaining, Cell Differentiation

Journal: The Journal of Cell Biology

Article Title: Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1

doi: 10.1083/jcb.201810058

Figure Lengend Snippet: Production and characterization of ETAA1 and TOPBP1-deficient cell lines. (A) Schematic of the ETAA1ΔAAD gene and protein. (B) HCT116 and HCT116 ETAA1ΔAAD cells were lysed and immunoblotted with ETAA1 antibodies. ETAA1 electrophoretic mobility is altered following CPT treatment. (C) Schematic of the TOPBP1-AID gene and protein. (D) HCT116 WT and TOPBP1-AID cells were treated with 500 µM IAA for the indicated times. Cells were lysed and immunoblotted with the indicated antibodies. Upper band is full-length TOPBP1-AID, and lower band is TOPBP1. (E–H) HCT116 WT, ETAA1ΔAAD, TOPBP1-AID, and ETAA1ΔAAD/TOPBP1-AID cells were left untreated (E and F) or treated (G and H) with 500 µM IAA. Viable cells numbers were measured by staining with trypan blue. Data points and error bars are mean and standard deviation of three experiments. (I) HCT116 WT, ETAA1ΔAAD, TOPBP1-AID, and ETAA1ΔAAD/TOPBP1-AID cells were left untreated or treated with 500 µM IAA for 2 h before examining cell cycle distribution by flow cytometry. Data points and error bars are the mean and standard deviation of three experiments. n.s., not significant. Molecular weight is given in kilodaltons.

Article Snippet: The following antibodies were used at 1:1,000 dilution for Western blotting or 1:200 for immunofluorescence unless otherwise noted: γH2AX (2577, Cell Signaling, rabbit), RPA32 (ab2175, 9H8, Abcam, mouse), pRPA32 S4/8 (A300-245A2, Bethyl, rabbit), pRPA32 S33 (A300-246A, Bethyl, rabbit), Flag (F-3165, M2, Sigma, mouse), ATR (SC-1887, N-19, Santa Cruz, goat), CHK1 (1:750, sc-8408, G4, Santa Cruz, mouse), CHK1 pS317 (2344S, Cell Signaling, rabbit), TOPBP1 (A300-111A-1, BL893, Bethyl, rabbit), BUB1 (A300-373A-M, Bethyl, rabbit), pH3 S10 (3377, Cell signaling, rabbit), Aurora B (A300-431A, Bethyl, rabbit), Aurora B pT232 (600-401-677, Roche, rabbit), α-tubulin (sc-5286, Santa Cruz, mouse), and anti-centromere antibodies (15-234-0001, Antibodies Inc., human).

Techniques: Staining, Standard Deviation, Flow Cytometry, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1

doi: 10.1083/jcb.201810058

Figure Lengend Snippet: ATR signaling in ETAA1 and TOPBP1-deficient cell lines. (A) HCT116 (WT), ETAA1ΔAAD, TOPBP1-AID, and ETAA1ΔAAD/TOPBP1-AID cells were treated with DMSO or IAA for 2 or 24 h and γH2AX induction measured by immunofluorescence imaging. Representative images and quantification of two independent experiments are show. Displayed is mean and SEM for each sample with >1,000 cells measured for each condition. Bar, 10 µm. (B) Cells were treated with 100 nM CPT for 4 h following a 2-h pretreatment with IAA or DMSO as indicated, then lysed and immunoblotted with the indicated antibodies. Molecular weight is given in kilodaltons.

Article Snippet: The following antibodies were used at 1:1,000 dilution for Western blotting or 1:200 for immunofluorescence unless otherwise noted: γH2AX (2577, Cell Signaling, rabbit), RPA32 (ab2175, 9H8, Abcam, mouse), pRPA32 S4/8 (A300-245A2, Bethyl, rabbit), pRPA32 S33 (A300-246A, Bethyl, rabbit), Flag (F-3165, M2, Sigma, mouse), ATR (SC-1887, N-19, Santa Cruz, goat), CHK1 (1:750, sc-8408, G4, Santa Cruz, mouse), CHK1 pS317 (2344S, Cell Signaling, rabbit), TOPBP1 (A300-111A-1, BL893, Bethyl, rabbit), BUB1 (A300-373A-M, Bethyl, rabbit), pH3 S10 (3377, Cell signaling, rabbit), Aurora B (A300-431A, Bethyl, rabbit), Aurora B pT232 (600-401-677, Roche, rabbit), α-tubulin (sc-5286, Santa Cruz, mouse), and anti-centromere antibodies (15-234-0001, Antibodies Inc., human).

Techniques: Immunofluorescence, Imaging, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1

doi: 10.1083/jcb.201810058

Figure Lengend Snippet: Quantitative phosphoproteomics identifies ETAA1- and TOPBP1-dependent phosphorylation sites. (A) Schematic of SILAC-phosphoproteomics approach. (B) Scatter plot of phosphoproteomic data. Each point represents a phosphosite, and the x and y value corresponds to the log 2 transformed ratios of ETAA1- and TOPBP1-dependent phosphorylation events. Higher ratio values correspond to more phosphorylation in WT cells than in ETAA1ΔAAD or TOPBP1-AID cells. (C and D) Number of phosphosites (C) and phosphoproteins (D) dependent on ETAA1, TOPBP1, or both ATR activators. (E) Diagram of ATR signaling pathway with preferred phosphorylation motifs of ATR and CHK1. (F and G) Abundance of ATR and CHK1 motifs in ETAA1- and TOPBP1-dependent phosphorylation sites.

Article Snippet: The following antibodies were used at 1:1,000 dilution for Western blotting or 1:200 for immunofluorescence unless otherwise noted: γH2AX (2577, Cell Signaling, rabbit), RPA32 (ab2175, 9H8, Abcam, mouse), pRPA32 S4/8 (A300-245A2, Bethyl, rabbit), pRPA32 S33 (A300-246A, Bethyl, rabbit), Flag (F-3165, M2, Sigma, mouse), ATR (SC-1887, N-19, Santa Cruz, goat), CHK1 (1:750, sc-8408, G4, Santa Cruz, mouse), CHK1 pS317 (2344S, Cell Signaling, rabbit), TOPBP1 (A300-111A-1, BL893, Bethyl, rabbit), BUB1 (A300-373A-M, Bethyl, rabbit), pH3 S10 (3377, Cell signaling, rabbit), Aurora B (A300-431A, Bethyl, rabbit), Aurora B pT232 (600-401-677, Roche, rabbit), α-tubulin (sc-5286, Santa Cruz, mouse), and anti-centromere antibodies (15-234-0001, Antibodies Inc., human).

Techniques: Phospho-proteomics, Multiplex sample analysis, Transformation Assay

Journal: The Journal of Cell Biology

Article Title: Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1

doi: 10.1083/jcb.201810058

Figure Lengend Snippet: TOPBP1-dependent phosphorylation GO analysis. (A) Functional GO network displaying grouping of GO terms enriched in TOPBP1-dependent phosphoproteins. Phosphoproteins and their corresponding GO terms were assigned functional groups after iterative merging of groups containing 50% of the same proteins. Each node in the network represents a GO term with a P value <0.05. A larger node size corresponds to more significant enrichment. Functional groups are indicated by color and represented in colored text by the most significant term in that group. Nodes with multiple colors belong to multiple groups. (B) Table displaying the 10 most highly enriched GO groups. The first term for each group represents the GO term with the highest significant enrichment. The second term for each group had the highest percentage of associated proteins for that GO group.

Article Snippet: The following antibodies were used at 1:1,000 dilution for Western blotting or 1:200 for immunofluorescence unless otherwise noted: γH2AX (2577, Cell Signaling, rabbit), RPA32 (ab2175, 9H8, Abcam, mouse), pRPA32 S4/8 (A300-245A2, Bethyl, rabbit), pRPA32 S33 (A300-246A, Bethyl, rabbit), Flag (F-3165, M2, Sigma, mouse), ATR (SC-1887, N-19, Santa Cruz, goat), CHK1 (1:750, sc-8408, G4, Santa Cruz, mouse), CHK1 pS317 (2344S, Cell Signaling, rabbit), TOPBP1 (A300-111A-1, BL893, Bethyl, rabbit), BUB1 (A300-373A-M, Bethyl, rabbit), pH3 S10 (3377, Cell signaling, rabbit), Aurora B (A300-431A, Bethyl, rabbit), Aurora B pT232 (600-401-677, Roche, rabbit), α-tubulin (sc-5286, Santa Cruz, mouse), and anti-centromere antibodies (15-234-0001, Antibodies Inc., human).

Techniques: Phospho-proteomics, Functional Assay

Journal: The Journal of Cell Biology

Article Title: Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1

doi: 10.1083/jcb.201810058

Figure Lengend Snippet: ETAA1 regulates phosphoproteins at kinetochores and spindles. (A) Flag-ETAA1 was exogenously expressed in 293T cells, immunopurified, and copurifying proteins were identified by mass spectrometry. Shown are the peptide counts for two experiments with two negative controls and two Flag purifications from parental cells lacking Flag-ETAA1. (B) HCT116 cells with or without stable expression of Flag-ETAA1 were treated with 100 nM taxol for 2 h and fixed, and Flag/BUB1 PLA signals were measured in mitotic and interphase cells. Bar, 5 µm. Quantification is the mean and standard deviation of three independent experiments in which at least 100 cells were analyzed for each condition. Significance was determined by ANOVA with a Dunnett multiple comparison post-test. (C) Diagram depicting the kinetochore- and spindle-associated phosphoproteins and sites detected in the ETAA1-dependent phosphoproteome. Each large gray circle contains the protein name with the smaller surrounding circles containing the observed phosphosites. The inner and outer phosphosite circle colors indicates the quantitative dependency on ETAA1 and TOPBP1, respectively.

Article Snippet: The following antibodies were used at 1:1,000 dilution for Western blotting or 1:200 for immunofluorescence unless otherwise noted: γH2AX (2577, Cell Signaling, rabbit), RPA32 (ab2175, 9H8, Abcam, mouse), pRPA32 S4/8 (A300-245A2, Bethyl, rabbit), pRPA32 S33 (A300-246A, Bethyl, rabbit), Flag (F-3165, M2, Sigma, mouse), ATR (SC-1887, N-19, Santa Cruz, goat), CHK1 (1:750, sc-8408, G4, Santa Cruz, mouse), CHK1 pS317 (2344S, Cell Signaling, rabbit), TOPBP1 (A300-111A-1, BL893, Bethyl, rabbit), BUB1 (A300-373A-M, Bethyl, rabbit), pH3 S10 (3377, Cell signaling, rabbit), Aurora B (A300-431A, Bethyl, rabbit), Aurora B pT232 (600-401-677, Roche, rabbit), α-tubulin (sc-5286, Santa Cruz, mouse), and anti-centromere antibodies (15-234-0001, Antibodies Inc., human).

Techniques: Mass Spectrometry, Expressing, Standard Deviation, Comparison, Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1

doi: 10.1083/jcb.201810058

Figure Lengend Snippet: ETAA1 activation of ATR is required for proper chromosome alignment and a fully functional SAC. (A and B) HCT116 WT, ETAA1ΔAAD, and TOPBP1-AID cells expressing GFP-H2B for chromatin visualization were examined by live cell imaging and scored for defects during mitosis. ATRi or IAA was added 1.5 h before beginning imaging. Representative time-lapse images are shown in A. White arrows indicate anaphase bridges and misaligned or lagging chromosomes. (B) Quantification of mitotic defects from live cell imaging experiments. Total number of cells examined for each cell type from three independent experiments is indicated in parentheses. (C) Representative immunofluorescence images of normal and misaligned chromosomes during metaphase. (D) HCT116 WT, ETAA1ΔAAD, and TOPBP1-AID cells were arrested for 16 h with CDK1i, treated with IAA or ATRi for 2 h, released from CDK1i, and fixed. Metaphase cells were scored for the number of misaligned chromosomes. (E) U2OS WT, ΔETAA1, and ΔETAA1 cells stably expressing ETAA1 were examined for chromosome misalignment as in D. D and E display the mean and standard deviation of three independent experiments in which at least 100 metaphases were examined per condition. (F) Synchronized HCT116 cells expressing H2B-GFP were released from a double thymidine block, and taxol, ATRi, or CHK1i were added 1.5 h before starting imaging. (G) Same as in F, but HCT116 WT and two clones of ETAA1ΔAAD and TOPBP1-AID cells were examined for ability to sustain mitotic arrest during taxol treatment. (F and G) The total number of cells examined for each cell type from three independent experiments is listed in parentheses. Bars: 5 µm (A and C).

Article Snippet: The following antibodies were used at 1:1,000 dilution for Western blotting or 1:200 for immunofluorescence unless otherwise noted: γH2AX (2577, Cell Signaling, rabbit), RPA32 (ab2175, 9H8, Abcam, mouse), pRPA32 S4/8 (A300-245A2, Bethyl, rabbit), pRPA32 S33 (A300-246A, Bethyl, rabbit), Flag (F-3165, M2, Sigma, mouse), ATR (SC-1887, N-19, Santa Cruz, goat), CHK1 (1:750, sc-8408, G4, Santa Cruz, mouse), CHK1 pS317 (2344S, Cell Signaling, rabbit), TOPBP1 (A300-111A-1, BL893, Bethyl, rabbit), BUB1 (A300-373A-M, Bethyl, rabbit), pH3 S10 (3377, Cell signaling, rabbit), Aurora B (A300-431A, Bethyl, rabbit), Aurora B pT232 (600-401-677, Roche, rabbit), α-tubulin (sc-5286, Santa Cruz, mouse), and anti-centromere antibodies (15-234-0001, Antibodies Inc., human).

Techniques: Activation Assay, Functional Assay, Expressing, Live Cell Imaging, Imaging, Immunofluorescence, Stable Transfection, Standard Deviation, Blocking Assay, Clone Assay

Journal: The Journal of Cell Biology

Article Title: Quantitative phosphoproteomics reveals mitotic function of the ATR activator ETAA1

doi: 10.1083/jcb.201810058

Figure Lengend Snippet: Models for ATR activation. (A) ATR is primarily activated by TOPBP1 in response to replication stress leading to phosphorylation of hundreds of proteins. ETAA1 contributes to ATR activation in response to replication stress, but regulates a smaller fraction of ATR substrates. (B) ETAA1 activates ATR localized to centromeric regions promoting full Aurora B activity, correct chromosome alignment, and a fully functional SAC.

Article Snippet: The following antibodies were used at 1:1,000 dilution for Western blotting or 1:200 for immunofluorescence unless otherwise noted: γH2AX (2577, Cell Signaling, rabbit), RPA32 (ab2175, 9H8, Abcam, mouse), pRPA32 S4/8 (A300-245A2, Bethyl, rabbit), pRPA32 S33 (A300-246A, Bethyl, rabbit), Flag (F-3165, M2, Sigma, mouse), ATR (SC-1887, N-19, Santa Cruz, goat), CHK1 (1:750, sc-8408, G4, Santa Cruz, mouse), CHK1 pS317 (2344S, Cell Signaling, rabbit), TOPBP1 (A300-111A-1, BL893, Bethyl, rabbit), BUB1 (A300-373A-M, Bethyl, rabbit), pH3 S10 (3377, Cell signaling, rabbit), Aurora B (A300-431A, Bethyl, rabbit), Aurora B pT232 (600-401-677, Roche, rabbit), α-tubulin (sc-5286, Santa Cruz, mouse), and anti-centromere antibodies (15-234-0001, Antibodies Inc., human).

Techniques: Activation Assay, Phospho-proteomics, Activity Assay, Functional Assay

Journal: Journal of Traditional Chinese Medicine

Article Title: Saponin Ⅰ from Shuitianqi ( Rhizoma Schizocapasae Plantagineae ) inhibits metastasis by negatively regulating the transforming growth factor-β1/Smad7 network and epithelial-mesenchymal transition in the intrahepatic metastasis Bagg’s Albino/c mouse model

doi: 10.19852/j.cnki.jtcm.2024.04.006

Figure Lengend Snippet: A: western blotting was used to detect the expression of N-cadherin, E-cadherin and Vimentin proteins. A1: model group; A2: cisplatin group; A3: SSPH Ⅰ (25 mg/kg) group; A4: SSPH Ⅰ (50 mg/kg) group; A5: SSPH Ⅰ (75 mg/kg) group. B: quantitative analysis of N-cadherin, E-cadherin and Vimentin proteins expression. C: immunohistochemical assay was used to detect the expression of E-cadherin protein in model group, cisplatin group and SSPH Ⅰ groups. C1: model group; C2: cisplatin group; C3: SSPH Ⅰ (25 mg/kg) group; C4: SSPH Ⅰ (50 mg/kg) group; C5: SSPH Ⅰ (75 mg/kg) group. magnification at ×200; scale bar, 20 μm, dyeing method of all pictures are the immunohistochemical DAB method. D: immunohistochemical assay was used to detect the expression of N-cadherin protein in model group, cisplatin group and SSPH Ⅰ groups. D1: model group; D2: cisplatin group; D3: SSPH Ⅰ (25 mg/kg) group; E4: SSPH Ⅰ (50 mg/kg) group; D5: SSPH Ⅰ (75 mg/kg) group. magnification at ×200; scale bar, 20 μm, dyeing method of all pictures are the immunohistochemical DAB method. E: immunohistochemical assay was used to detect the expression of Vimentin protein in model group, cisplatin group and SSPH Ⅰ groups. E1: model group; E2: cisplatin group; E3: SSPH Ⅰ (25 mg/kg) group; E4: SSPH Ⅰ (50 mg/kg) group; E5: SSPH Ⅰ (75 mg/kg) group. magnification at ×200; scale bar, 20 μm, dyeing method of all pictures are the immunohistochemical DAB method. F: quantitative analysis of N-cadherin, E-cadherin and Vimentin proteins expression. model group: free diet + gavage with equal amount of normal saline per day; cisplatin group: model group + intraperitoneal injection with equal amount of cisplatin 2 mg/kg every other day; SSPH Ⅰ (25 mg/kg) group: model group + gavage with equal amount of SSPH Ⅰ 25 mg/kg per day; SSPH Ⅰ (50 mg/kg) group: model group + gavage with equal amount of SSPH Ⅰ 50 mg/kg per day; SSPH Ⅰ (75 mg/kg) group: model group + gavage with equal amount of SSPH Ⅰ 75 mg/kg per day. SSPH Ⅰ: Saponin I from Shuitianqi (Rhizoma Schizocapasae Plantagineae); EMT: epithelial-mesenchymal transition; DAB: diaminobenzidine. All data was measured by one-way analysis, and Newman-Keuls test was performed for inter-group comparisons. All data was presented as mean ± standard deviation (n = 3). aP < 0.05, bP < 0.001 compared to the model group.

Article Snippet: Subsequently, sections were incubated in 5% BSA blocking fluid for 1 h, with subsequent ON incubation in primary antibodies at 4 ℃: E-cadherin, N-cadherin, Vimentin, TGF-β1(PTG, 21898-1-AP), Smad7, MMP-2, MMP-9, CD31, CD34 (Sino Biological Inc., 50589-T24), and VEGF (all dilution 1:200).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Saline, Injection, Standard Deviation

Journal: PLOS One

Article Title: Regulation of tension-dependent localization of LATS1 and LATS2 to adherens junctions

doi: 10.1371/journal.pone.0342107

Figure Lengend Snippet: (A) Full-length LATS2, LATS2-LATCH and LATS2-LATCH-4mut, were tested for binding to LIMD1 by co-immunoprecipitation. Lysates were prepared from HEK293A cells separately transfected with GFP-tagged LATS2, LATS2-LATCH, LATS2-LATCH-4mut, or LIMD1-V5. Lysates from cells expressing LIMD1-V5 were mixed separately with those expressing the different GFP-LATS2/LATCH constructs, and anti-LIMD1 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for LATS2/LATCH (GFP) and LIMD1 (V5). (B) Live-cell imaging of MCF10A cells stably expressing GFP tagged LATS2-LATCH or LATS2-LATCH-4mut. (C) Live-cell imaging of MCF10A cells stably expressing GFP tagged LATS2-LATCH treated with (Blebbistatin) or without (DMSO) Blebbistatin.

Article Snippet: Immunoprecipitation was carried out using 1 ug of rabbit anti-LIMD1 (Novus biologicals, NBP2–56448), mouse anti-Myc (Cell Signaling, 2276) or mouse anti-FLAG antibody (Sigma-Aldrich, F1804) coupled to 50 uL of Dynabeads at 4°C with gentle shaking for 1 hour.

Techniques: Binding Assay, Immunoprecipitation, Transfection, Expressing, Construct, Control, Western Blot, Live Cell Imaging, Stable Transfection

Journal: PLOS One

Article Title: Regulation of tension-dependent localization of LATS1 and LATS2 to adherens junctions

doi: 10.1371/journal.pone.0342107

Figure Lengend Snippet: (A-B) LIMD1-KO MCF10A cells stably expressing GFP tagged wild-type LIMD1 (WT) or LIMD1 strain insensitive mutants (F512A, F575A, and Y646A, see also .) were established by lentiviral transduction and imaged using fixed and live-cell imaging. (A) The indicated cell lines were stained using anti-LATS1 and anti-TRIP6 antibodies. Merged images show LATS1 (red), TRIP6 (green) and DNA (blue). (B) The indicated cell lines from part (A) were imaged live for GFP fluorescence. (C) Quantification of the LATS1 junction to cytoplasm mean intensity ratio. Error bars represent the standard deviation. ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the plot (mean ± SD; n = 5; ****P < 0.0001). (D-E) LATS1/2 were tested for their ability to bind to strain insensitive mutants of LIMD1 by co-immunoprecipitation. (D) LATS2 was tested for its ability to bind to wild-type (WT) and mechanical strain insensitive mutants (F512A, F575A or Y646A) of LIMD1 by co-immunoprecipitation. V5-tagged WT and mutants of LIMD1 and LATS2-FLAG were separately transfected in HEK293A cells. HEK293A lysates from cells transfected with V5-tagged WT and mutants of LIMD1 were combined with LATS2-FLAG lysates. Anti-LIMD1 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for V5-tagged WT and mutants of LIMD1 (V5) and LATS2-FLAG (LATS2). (E) LATS1 was tested for binding to wild-type (WT) and mechanical strain insensitive mutants (F512A, F575A or Y646A) of LIMD1 by co-immunoprecipitation. V5-tagged WT and mutants of LIMD1, and LATS1-3xMyc were separately transfected in HEK293A cells. HEK293A lysates from cells transfected with V5-tagged LIMD1 variants were combined with LATS1-3xMyc lysates. Anti-Myc or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for V5-tagged WT and mutants of LIMD1 (LIMD1) and LATS1-3xMyc (LATS1). (F) Quantification of relative amounts of LATS2 (normalized to wild-type LIMD1 in the IP fraction) immunoprecipitated by wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants in part (D) . ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the graph (mean ± SD; n = 3; ***P = 0.001, ****P < 0.0001). (G) Quantification of relative amounts of wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants (normalized to wild-type LIMD1 in the input fraction) immunoprecipitated by LATS1-3xMyc from part (E) . ANOVA statistical comparisons between wild-type LIMD1 and mechanical strain insensitive LIMD1 mutants are indicated above the graph (mean ± SD; n = 3; *P < 0.05, ***P = 0.001).

Article Snippet: Immunoprecipitation was carried out using 1 ug of rabbit anti-LIMD1 (Novus biologicals, NBP2–56448), mouse anti-Myc (Cell Signaling, 2276) or mouse anti-FLAG antibody (Sigma-Aldrich, F1804) coupled to 50 uL of Dynabeads at 4°C with gentle shaking for 1 hour.

Techniques: Stable Transfection, Expressing, Transduction, Live Cell Imaging, Staining, Fluorescence, Standard Deviation, Immunoprecipitation, Transfection, Control, Western Blot, Binding Assay

Journal: PLOS One

Article Title: Regulation of tension-dependent localization of LATS1 and LATS2 to adherens junctions

doi: 10.1371/journal.pone.0342107

Figure Lengend Snippet: (A) Live-cell imaging of MCF10A cells stably expressing GFP tagged LATS1 or LATS2 N-terminal regions (aa 1-635 in LATS1, aa 1-598 in LATS2) with and without (∆LATCH) the LATCH regions (aa 472-520 in LATS1, aa 418-466 in LATS2) as indicated. (B) AlphaFold2 model showing the three tandem LIM domains of LIMD1 (orange) and two regions of LATS2 (green) that are predicted to interact (LATS-LATCH and the Helical Hairpin). (C) Multiple sequence alignments of LATS1/2 from the indicated species showing the conserved LATCH region. (D-E) The requirement of the conserved LATCH sequence of LATS1/2 to bind with LIMD1 was tested by co-immunoprecipitation. (D) Full-length LATS2 (LATS2) or LATS2 with either the LATCH deleted (LATS2-∆LATCH, aa 418-466 deleted) or with the helical hairpin region deleted (LATS2-∆HH, aa 599-667 deleted) tagged to FLAG and (E) Full-length LATS1 (LATS1) or LATS1 with the LATCH deleted (LATS1-∆LATCH, aa 472-520 deleted) tagged with 3xMyc were tested for binding to LIMD1 by co-immunoprecipitation. FLAG-LATS2 variants, LATS1-3xMyc variants, and LIMD1-V5 were separately transfected in HEK293 cells. HEK293A cell lysates transfected with either FLAG-LATS2 variants (D) or LATS1-3xMyc variants (E) were combined with V5-tagged LIMD1. Anti-LIMD1 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by Western blotting for LATS2 variants (FLAG) (D) , LATS1 variants (Myc) (E) and LIMD1 (V5).

Article Snippet: Immunoprecipitation was carried out using 1 ug of rabbit anti-LIMD1 (Novus biologicals, NBP2–56448), mouse anti-Myc (Cell Signaling, 2276) or mouse anti-FLAG antibody (Sigma-Aldrich, F1804) coupled to 50 uL of Dynabeads at 4°C with gentle shaking for 1 hour.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Sequencing, Immunoprecipitation, Binding Assay, Transfection, Control, Western Blot

Journal: The Journal of Cell Biology

Article Title: Coupling of Cdc20 inhibition and activation by BubR1

doi: 10.1083/jcb.202012081

Figure Lengend Snippet: PP2A-B56 and PP2A-B55 are phosphatases for Cdc20 T70. (A) Cells treated with a control RNAi oligo or a pool of B56 RNAi oligoes targeting all isoforms were arrested in nocodazole and Cdc20 immunopurified. Samples were analyzed by Western blot for Cdc20 T70p and APC3 T447p as well as APC3 and BubR1. The degree of Cdc20 T70p and APC3 T447p was determined by normalizing to pan-Cdc20 or pan-APC3 signals. (B and C) A nocodazole lysate was prepared and treated with the indicated phosphatase inhibitors (see also ) for 5 or 15 min as indicated. Cdc20 or APC4 was immunopurified and the level of Cdc20 T70p and APC3 T447p determined. For all experiments, three independent experiments were conducted and quantified. Ctrl, control.

Article Snippet: The following antibodies were used at the indicated dilutions for Western blot: Cdc20 mouse monoclonal antibody (1:1,000; sc-13162; Santa Cruz Biotechnology; clone E7, indicated as #1), Cdc20 mouse monoclonal antibody (MAB3775; Millipore; clone AR12, indicated as #2), Cdc20 T70 and T59 phosphorylation-specific antibodies rabbit polyclonal antibody [raised against peptides CSKVQT(Tp)PSKPG and CRTPGR(Tp)PGKSS; Moravian Biotechnology; reference 22], APC3 T447p rabbit polyclonal [raised against Peptides CGKISTI(Tp)PQIQAF; Moravian Biotechnology], anti-phospho-histone H3 (Ser 10) rabbit polyclonal antibody (1:1,000; 06–570; Millipore), APC4 mouse monoclonal antibody (1:500; Moravian Biotechnology; clone CIV 1.1), APC3 mouse monoclonal antibody (1:500; Moravian Biotechnology), Mad2 rabbit polyclonal antibody (1:500; A300-300A; Bethyl Laboratories), Bub3 mouse monoclonal antibody (1:500; 611731; BD Biosciences), BubR1 rabbit polyclonal antibody (1:1,000; A300-995A; Bethyl Laboratories), and BubR1 mouse monoclonal antibody (1:500; Biotech Research and Innovation Centers; raised against TPR domain).

Techniques: Control, Western Blot

Journal: The Journal of Cell Biology

Article Title: Coupling of Cdc20 inhibition and activation by BubR1

doi: 10.1083/jcb.202012081

Figure Lengend Snippet: Cdc20 T70 is dephosphorylated by BubR1 bound PP2A-B56. (A) Schematic indicating Cdc20 and PP2A-B56 binding to BubR1. The mutations in BubR1 2A are indicated. (B) Stable cell lines expressing Venus-BubR1 WT or 2A were depleted of BubR1 by RNAi and arrested in mitosis with nocodazole. Cdc20 was immunopurified from the cells and the degree of Cdc20 T70p and APC3 T447p determined. Samples were compared with similar samples from the parental cells not treated with BubR1 RNAi. (C) Cdc20, BubR1, and APC4 were immunopurified from nocodazole arrested cells and the level of Cdc20 T70p and APC3 T447p determined. The signal from the phosphospecific antibody was normalized to the pan-specific antibody for Cdc20, and for APC3 T447p the signal was normalized to APC4. For Cdc20, two different antibodies were used. For all experiments, at least three independent experiments were conducted and quantified. Ctrl, control.

Article Snippet: The following antibodies were used at the indicated dilutions for Western blot: Cdc20 mouse monoclonal antibody (1:1,000; sc-13162; Santa Cruz Biotechnology; clone E7, indicated as #1), Cdc20 mouse monoclonal antibody (MAB3775; Millipore; clone AR12, indicated as #2), Cdc20 T70 and T59 phosphorylation-specific antibodies rabbit polyclonal antibody [raised against peptides CSKVQT(Tp)PSKPG and CRTPGR(Tp)PGKSS; Moravian Biotechnology; reference 22], APC3 T447p rabbit polyclonal [raised against Peptides CGKISTI(Tp)PQIQAF; Moravian Biotechnology], anti-phospho-histone H3 (Ser 10) rabbit polyclonal antibody (1:1,000; 06–570; Millipore), APC4 mouse monoclonal antibody (1:500; Moravian Biotechnology; clone CIV 1.1), APC3 mouse monoclonal antibody (1:500; Moravian Biotechnology), Mad2 rabbit polyclonal antibody (1:500; A300-300A; Bethyl Laboratories), Bub3 mouse monoclonal antibody (1:500; 611731; BD Biosciences), BubR1 rabbit polyclonal antibody (1:1,000; A300-995A; Bethyl Laboratories), and BubR1 mouse monoclonal antibody (1:500; Biotech Research and Innovation Centers; raised against TPR domain).

Techniques: Binding Assay, Stable Transfection, Expressing, Control

Journal: The Journal of Cell Biology

Article Title: Coupling of Cdc20 inhibition and activation by BubR1

doi: 10.1083/jcb.202012081

Figure Lengend Snippet: Cdk1 phoshorylation of Cdc20 is required for an efficient mitotic arrest in response to microtubule poisons. (A) Live cell imaging of Hela cells depleted of endogenous Cdc20 and transiently expressing untagged Cdc20 WT, 3DD, or 3AA in the presence of nocadazole (30 ng/ml). Percentage of cells that exit mitosis during the course of imaging from three independent experiments (mean and SD indicated). (B) As in A, time from nuclear envelope breakdown (NEBD) to mitotic exit or end of filming from three independent experiments (each dot represents a single cell, red line indicates mean and SD, n values are shown in the graph and represent single cells analyzed per condition; statistical analysis with Mann-Whitney test; ****P > 0.0001). (C) Live cell imaging of stable cell lines that were depleted of endogenous Cdc20 and expressing indicated YFP-tagged Cdc20 proteins in the presence of nocodazole (30 ng/ml). The R132A mutation prevents Mad2 binding, while the 4A mutations prevent BubR1 binding. Time from NEBD to mitotic exit (each dot represents a single cell, red line indicates mean and standard deviation, n values are shown in the graph and represent single cells analyzed per condition, statistical analysis with Mann-Whitney test; ****P > 0.0001). (D) Model: MCC-bound BubR1 is preferentially dephosphorylated by PP2A-B56, whereas free Cdc20 is phosphorylated by Cdk1.

Article Snippet: The following antibodies were used at the indicated dilutions for Western blot: Cdc20 mouse monoclonal antibody (1:1,000; sc-13162; Santa Cruz Biotechnology; clone E7, indicated as #1), Cdc20 mouse monoclonal antibody (MAB3775; Millipore; clone AR12, indicated as #2), Cdc20 T70 and T59 phosphorylation-specific antibodies rabbit polyclonal antibody [raised against peptides CSKVQT(Tp)PSKPG and CRTPGR(Tp)PGKSS; Moravian Biotechnology; reference 22], APC3 T447p rabbit polyclonal [raised against Peptides CGKISTI(Tp)PQIQAF; Moravian Biotechnology], anti-phospho-histone H3 (Ser 10) rabbit polyclonal antibody (1:1,000; 06–570; Millipore), APC4 mouse monoclonal antibody (1:500; Moravian Biotechnology; clone CIV 1.1), APC3 mouse monoclonal antibody (1:500; Moravian Biotechnology), Mad2 rabbit polyclonal antibody (1:500; A300-300A; Bethyl Laboratories), Bub3 mouse monoclonal antibody (1:500; 611731; BD Biosciences), BubR1 rabbit polyclonal antibody (1:1,000; A300-995A; Bethyl Laboratories), and BubR1 mouse monoclonal antibody (1:500; Biotech Research and Innovation Centers; raised against TPR domain).

Techniques: Live Cell Imaging, Expressing, Imaging, Single Cell, MANN-WHITNEY, Stable Transfection, Mutagenesis, Binding Assay, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Coupling of Cdc20 inhibition and activation by BubR1

doi: 10.1083/jcb.202012081

Figure Lengend Snippet: Cdk1 phoshorylation of Cdc20 is required for an efficient mitotic arrest in response to microtubule poisons. (A) Live cell imaging of Hela cells depleted of endogenous Cdc20 and transiently expressing untagged Cdc20 WT, 3DD, or 3AA in the presence of nocadazole (30 ng/ml). Percentage of cells that exit mitosis during the course of imaging from three independent experiments (mean and SD indicated). (B) As in A, time from nuclear envelope breakdown (NEBD) to mitotic exit or end of filming from three independent experiments (each dot represents a single cell, red line indicates mean and SD, n values are shown in the graph and represent single cells analyzed per condition; statistical analysis with Mann-Whitney test; ****P > 0.0001). (C) Live cell imaging of stable cell lines that were depleted of endogenous Cdc20 and expressing indicated YFP-tagged Cdc20 proteins in the presence of nocodazole (30 ng/ml). The R132A mutation prevents Mad2 binding, while the 4A mutations prevent BubR1 binding. Time from NEBD to mitotic exit (each dot represents a single cell, red line indicates mean and standard deviation, n values are shown in the graph and represent single cells analyzed per condition, statistical analysis with Mann-Whitney test; ****P > 0.0001). (D) Model: MCC-bound BubR1 is preferentially dephosphorylated by PP2A-B56, whereas free Cdc20 is phosphorylated by Cdk1.

Article Snippet: The following antibodies were used at the indicated dilutions for Western blot: Cdc20 mouse monoclonal antibody (1:1,000; sc-13162; Santa Cruz Biotechnology; clone E7, indicated as #1), Cdc20 mouse monoclonal antibody (MAB3775; Millipore; clone AR12, indicated as #2), Cdc20 T70 and T59 phosphorylation-specific antibodies rabbit polyclonal antibody [raised against peptides CSKVQT(Tp)PSKPG and CRTPGR(Tp)PGKSS; Moravian Biotechnology; reference 22], APC3 T447p rabbit polyclonal [raised against Peptides CGKISTI(Tp)PQIQAF; Moravian Biotechnology], anti-phospho-histone H3 (Ser 10) rabbit polyclonal antibody (1:1,000; 06–570; Millipore), APC4 mouse monoclonal antibody (1:500; Moravian Biotechnology; clone CIV 1.1), APC3 mouse monoclonal antibody (1:500; Moravian Biotechnology), Mad2 rabbit polyclonal antibody (1:500; A300-300A; Bethyl Laboratories), Bub3 mouse monoclonal antibody (1:500; 611731; BD Biosciences), BubR1 rabbit polyclonal antibody (1:1,000; A300-995A; Bethyl Laboratories), and BubR1 mouse monoclonal antibody (1:500; Biotech Research and Innovation Centers; raised against TPR domain).

Techniques: Live Cell Imaging, Expressing, Imaging, Single Cell, MANN-WHITNEY, Stable Transfection, Mutagenesis, Binding Assay, Standard Deviation