Journal: International journal of biological macromolecules

Article Title: Disulfide bond and crosslinking analyses reveal inter-domain interactions that contribute to the rigidity of placental malaria VAR2CSA structure and formation of CSA binding channel.

doi: 10.1016/j.ijbiomac.2022.11.258

Figure Lengend Snippet: Fig. 8. The binding capacity of full-length VAR2CSA ectodomain is lower than the ectodomain lacking NTS. The binding of NTS-DBL6ε and DBL1x-DBL6ε to CSPG was measured in triplicates by ELISA. The wells of 96-well microtiter plates were coated with CSPG (200 ng/ml) and blocked with BSA and then incubated with 1:2 serially diluted solutions of NTS-DBL6ε or DBL1x-DBL6ε. The levels of bound proteins were measured using anti-cMyc antibody and HRP- conjugated secondary antibody. Data are a representative of three independent experiments using different batches of purified proteins. KD values were determined by plotting OD vs protein concentration using GraphPad Prism v9.4.1. KD ± SE: 41.5 ± 3.9 (DBBL1x-DBL6ε); 59.9 ± 6.8 (NTS-DBL6ε).

Article Snippet: Unbound proteins in the coated wells were removed by washing with 100 μl of PBST and the plates were incubated with 1:1000 diluted mouse anti-cMyc monoclonal antibody (Cat No. NB600-302, NOVUS Biologicals, Centennial, CO), and washed three times with 100 μl PBST.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Purification, Protein Concentration

Journal: bioRxiv

Article Title: WNK kinase regulates plasma membrane levels of the WNT inhibitor RNF43

doi: 10.1101/2025.10.08.681128

Figure Lengend Snippet: a Schematic of the biotinylation experiment coupled to Mass spectrometry analysis. b Volcano plot illustrating logarthmic fold changes of proteins identified in HRP- and APEX2-mediated biotinylation experiments. A total of 577 proteins (HRP-RNF43) and 183 proteins (RNF43-APEX2) met the threshold for statistical significance (≥ 2-fold change, p ≤ 0.05) and are denoted in red. Proteins with a ≥ 2-fold change but not statistically significant are shown in green, while proteins with statistically significant p -values ( p ≤ 0.05) but fold change < 2 are shown in blue. Fold changes and p -values were derived from triplicate experiments and corresponding controls using imputed data. c Heatmap of proteomic expression of HRP-RNF43 biotinylation interactors. Row z-scores represent the number of standard deviations each protein’s expression deviates from its mean across triplicate experiments. Red and blue indicate higher and lower expression levels, respectively. Differential expression of 21 HRP-RNF43 interactors is shown, with SORT1 and SORL1 highlighted in red text. d Heatmap of proteomic expression of 10 RNF43-APEX2 interactors based on duplicate control and triplicate experimental conditions. The interactor WNK1 is highlighted in red text. e SNAP-labeling of SNAP-Fzd5 expressing cells. In cells co-transfected with RNF43, Fzd5 rapidly relocates into cytosolic vesicles (puncta). However, treatment with the clathrin inhibitor PITSTOP partially suppresses Fzd5 endocytosis. Nystatin, an inhibitor of caveolin-dependent endocytosis, has no effect on RNF43-dependent Fzd5 endocytosis. Scale bar represents 50 μm. f, g Immunoprecipitation and Western blot analysis showing interactions between RNF43 and endogenous WNK1 and overexpressed SORTILIN. Unrelated IgG ( f ) and eGFP ( g ) were used as negative controls.

Article Snippet: The following plasmids were obtained from Addgene: rat myc-tagged WNK1 (38779); human His-6-,V5-tagged Wnk2 (24569); pCAG-HRP-TM (44441); pcDNA3 APEX2-NES (49386).

Techniques: Mass Spectrometry, Derivative Assay, Expressing, Quantitative Proteomics, Control, Labeling, Transfection, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: WNK kinase regulates plasma membrane levels of the WNT inhibitor RNF43

doi: 10.1101/2025.10.08.681128

Figure Lengend Snippet: a Lollipop plot of KEGG pathway enrichment among the 577 differentially expressed HRP-RNF43 proteins (fold change ≥ 2, p ≤ 0.05). The x-axis denotes the number of associated proteins per pathway; circle size reflects statistical significance (−log 10 FDR). The top seven pathways are shown, with Wnt signaling most enriched, followed by Cadherin and Integrin signaling. b KEGG pathway analysis of 183 RNF43-APEX2 differentially expressed proteins (fold change ≥ 2, p ≤ 0.05), highlighting the top seven pathways. Protein processing in the endoplasmic reticulum is the most significantly enriched. c UpSet plot of HRP-RNF43 proteins across oncology subtypes using only significant proteins (fold change ≥ 2, p ≤ 0.05). Red dots without connecting lines indicate disease-specific proteins, while connected dots denote overlap across multiple cancer types. Vertical line length corresponds to the number of shared subtypes. The y-axis indicates the number of proteins per cancer category. d Gene-concept network (cnetplot) illustrating connections between significant HRP-RNF43 proteins and malignancy-related pathways. Proteins are represented as dots, colored by fold change (white to red), with bolded labels indicating HRP-RNF43 interactors. Node proximity reflects strength of literature-based association, and node size indicates the number of associated proteins (cluster size). e, f IP experiments followed by Western blot analysis showing that RNF43 interacts with overexpressed WNK2 ( e ) or endogenous WNK1 ( f ). Note that Rspo1 treatment does not increase RNF43/WNK interaction. g IP experiment followed by Western blot analysis confirming RNF43 interaction with Sortilin-like1 (SorL1) protein. Treatment with Rspo1 does not have any effect on this interaction (lane 4).

Article Snippet: The following plasmids were obtained from Addgene: rat myc-tagged WNK1 (38779); human His-6-,V5-tagged Wnk2 (24569); pCAG-HRP-TM (44441); pcDNA3 APEX2-NES (49386).

Techniques: Western Blot