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

Journal: Cell reports

Article Title: ARHGEF3 Regulates Skeletal Muscle Regeneration and Strength through Autophagy

doi: 10.1016/j.celrep.2020.108594

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: pCMV-Myc-ARHGEF3 (XPLN)-L269E , , Addgene Plasmid #73376.

Techniques: Recombinant, Plasmid Preparation, Software, Imaging

Journal: PLoS Biology

Article Title: Role of Mitochondrial Dynamics in Neuronal Development: Mechanism for Wolfram Syndrome

doi: 10.1371/journal.pbio.1002511

Figure Lengend Snippet: (A) Neurons were transfected with plasmids expressing scrambled shRNA or Wfs1 shRNA, firefly luciferase constructs containing ATF6 or ATF4 binding sites or a XBP-1 splicing reporter, and Renilla luciferase. Firefly luciferase signal normalized to Renilla signal demonstrates a moderate increase in ATF6 and ATF4 reporter activity. (B) Positive control experiments in which the above-mentioned reporter systems and Renilla luciferase were co-transfected with ATF4, ATF6, or IRE1. (C) The mitochondrial fusion rate is reduced by Wfs1 shRNA and is restored by co-expressing wt HSPA5 ( p = 0.001 for interaction, two-way ANOVA). (D) ATPase-deficient HSPA5 mutant (T37G) but not peptide binding-deficient mutant (P495L) restores the fusion rate reduced by Wfs1 shRNA. (E) HSPA5 overexpression attenuates mitophagy activated by Wfs1 silencing ( p = 0.012 for interaction). (F–H) Activation of the primary ER stress pathways by overexpression of ATF6, ATF4, or IRE1 modulates neither fusion rate (F), mitochondrial length (G), nor mitophagy (H). (I–L) Silencing of ATF6 or ATF4 modulates neither fusion rate (I, J) nor mitophagy (K, L). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with respective control groups, or ### p < 0.001 compared with the Wfs1 shRNA-transfected control group and ns non-significant compared with the Wfs1 shRNA-transfected control group. Underlying data is shown in .

Article Snippet: ATeam (51958), ATF6 (11975), ATF6-GL3 (11976), ATF4 (26114), ATF4-luc (21850), WFS1 wt (13011), WFS1 P724L (13012), IRE1α (13009), D1ER (36325), D3cpv (36323), DRP1 K38A, EGFP-LC3 (24920), HSPA5 wt (27164), HSPA5 T37G (27165), HSPA5 P495L (27166), NRF2 (21555), ORAI1 (21638), PSD-95 (15463), and pAAV-hSyn-DsRedExpress (22907) were obtained from Addgene (Cambridge, MA).

Techniques: Transfection, Expressing, shRNA, Luciferase, Construct, Binding Assay, Activity Assay, Positive Control, Mutagenesis, Over Expression, Activation Assay, Control

Journal: bioRxiv

Article Title: Golgi Renaissance: the pivotal role of the largest Golgi protein giantin

doi: 10.1101/296517

Figure Lengend Snippet: ( A ) Confocal immunofluorescence images of giantin and ASGP-R in VA-13 cells: control, EtOH-treated cells, EtOH-treated cells and transfected with scramble, giantin, NMIIB, Rab6a siRNAs, and dominant negative (GDP-bound) Rab6a(T27N) followed by recovery. White boxes are enlarged pictures of ASGP-R presented at the right side. Arrowheads indicate ASPR-R punctae distributed at the periphery of cells. All confocal images acquired with same imaging parameters; bars, 10 μm. ( B ) Quantification of cells with compact Golgi in cells presented in A; n = 90 cells from three independent experiments, results expressed as a mean ± SD; *, p<0.001. ( C ) Giantin, NMIIB, and Rab6a W-B of lysates of VA-13 cells treated with corresponding siRNAs; β-actin was a loading control. ( D ) Transferrin, PIGR, and ASGP-R W-B of plasma membrane fractions isolated from VA-13 cells presented in A; samples were normalized to E-cadherin. ( E ) SNA lectin W-B of ASGPR-IP from the plasma membrane fractions isolated from VA-13 cells: control, EtOH-treated, and recovered from EtOH in absence or presence of giantin siRNAs. The input was normalized to the E-cadherin.

Article Snippet: PCMV-intron myc Rab6 T27N was a gift from Terry Hebert (Addgene plasmid # 46782) ( ).

Techniques: Immunofluorescence, Transfection, Dominant Negative Mutation, Imaging, Isolation