Journal: bioRxiv

Article Title: RBMX2: A Pivotal Regulator Linking Mycobacterium bovis Infection to Epithelial-Mesenchymal Transition and Lung Cancer Progression

doi: 10.1101/2025.04.15.648920

Figure Lengend Snippet: (A, B) Activation of the MAPK pathway-related protein and p65 protein were activated after RBMX2 knockout and WT EBL cells infected by M. bovis via WB. Data were relative to WT EBL cells with M. bovis infection. (C, D) Expression of tight junction-related proteins (ZO-1, CLDN-5, and OCLN) was assessed in RBMX2 knockout EBL cells treated with three p38/p65/JNK pathways activators after M. bovis infection via WB. Data were relative to RBMX2 knockout EBL cells untreated activators with M. bovis infection. (E, F) Evaluate the impact of three p38/p65/JNK pathways activators on the ratio of intercellular adhesion via cell adhesion assay. Data were relative to RBMX2 knockout EBL cells untreated activators with M. bovis infection. Scale bar: 20 μm. (G, H) Evaluate the silencing efficiency of siRNA on p65 protein expression and its impact on the expression of ZO-1, CLDN-5, and OCLN proteins through WB. Data were relative to SiRNA-NC in WT EBL cells with M. bovis infection. (I) Detect the correlation between p65 expression and ZO-1 in WT EBL cells after M. bovis infection via IF analysis. ZO-1 is stained with green fluorescence, p65 is stained with yellow fluorescence, and the nucleus is stained with blue fluorescence. Data were relative to SiRNA-NC in WT EBL cells with M. bovis infection. (J) The effect of p65 silencing on the invasive ability of M. bovis in WT EBL cells. Data were relative to SiRNA-NC in WT EBL cells with M. bovis infection. (K) The effect of RBMX2 on the nuclear translocation of p65 protein after M. bovis infection using WB. β-actin presents cytosol and Lamin A/C presents nucleus. Data were relative to RBMX2 knockout EBL cells after M. bovis infection. (L) The effect of RBMX2 on the nuclear translocation of p65 protein after BCG -infection using High-content real-time imaging. Using pCMV-EGFP-p65 plasmid transfect RBMX2 knockout and WT EBL cells. The nucleus is stained with blue fluorescence. Data were relative to WT EBL cells without BCG infection. (M, N, O) The impact of M. bovis on the adhesion, invasion, and intracellular survival of RBMX2 knockout and WT EBL cells through plate counting. Data were relative to WT EBL cells after M. bovis infection. (P, Q, R) The impact of M. bovis on the adhesion, invasion, and intracellular survival of Sh-NC and Sh-RBMX2 H1299 cells through plate counting. Data were relative to H1299 ShNC cells after M. bovis infection. One-way ANOVA and two-way ANOVA were used to determine the statistical significance of differences between different groups. Ns presents no significance, * presents p < 0.05, **presents p < 0.01, and *** presents p < 0.001 indicate statistically significant differences. Data were representative of at least three independent experiments.

Article Snippet: The following primary antibodies were utilized: ZO-1 (Bioss, China), OCLN (Proteintech, China), CLADN-5 (Proteintech, China), MMP-9 (Proteintech, China), E-cadherin (Proteintech, China), N-cadherin (Bioss, China), LaminA/C (Proteintech, China), β-actin (Bioss, China), p65 (CST, USA), p-p65 (CST, USA), and a MAPK-related antibody (CST, USA).

Techniques: Activation Assay, Knock-Out, Infection, Expressing, Cell Adhesion Assay, Staining, Fluorescence, Translocation Assay, Imaging, Plasmid Preparation

Journal: bioRxiv

Article Title: A conserved protein tyrosine phosphatase, PTPN-22, functions in diverse developmental processes in C. elegans

doi: 10.1101/2024.03.12.584557

Figure Lengend Snippet: (A) Schematic illustrating the proximity labeling study. The C terminus of PTPN-22 was fused to TurboID::3×-FLAG, leading to the biotinylation of proximal proteins. Proximal proteins are depicted in blue, with the resulting biotin modification highlighted in red, whereas proteins located outside the TurboID labeling radius (∼10 nm) are represented in gray. PTPN-22::TurboID or N2 control animals were cultured on plates, and subsequent protein extraction was carried out. Biotinylated proteins were pulled down using streptavidin-coated magnetic beads (orange), whereas non-biotinylated proteins were removed through washing steps. Enriched biotinylated proteins were subjected to on-bead digestion, followed by Data-Independent Acquisition (DIA) LC-MS/MS analysis. (B) Western blot (WB; left) shows the input fractions of representative N2 and PTPN-22::TurboID samples probed with streptavidin-HRP. Note additional bands in the PTPN-22::TurboID lysate versus the N2 control. The expression of PTPN-22::TurboID was visualized through an anti-FLAG western blot; antibodies against β-actin were used as a loading control. The pull-down fraction (IP, right) shows N2 and PTPN-22::TurboID samples probed with streptavidin-HRP after enriching for biotinylated proteins using streptavidin-coated beads. (C) Volcano plot highlighting proteins enriched (>2-fold and p-value <0.05) in PTPN-22::TurboID samples (red) versus N2 (blue). (D) Dot plots show the enrichment of PTPN-22 in PTPN-22::TurboID samples; error bars represent standard deviation. (E) KEGG pathway enrichment analysis was performed using ShinyGO 0.80, and the top 19 biological pathways based on fold enrichment are shown . (F) Venn diagram shows the overlap of enriched proteins between PTPN-22::TurboID samples and P granule proteins (S2 File) (G) The dot plot shows the brood size of individual worms in the indicated backgrounds

Article Snippet: Horseradish peroxidase(HRP)–conjugated streptavidin (streptavidin-HRP; Cat# 3999S) and HRP-conjugated rabbit monoclonal antibody against β-actin (Cat# 5125S) were obtained from Cell Signaling Technology.

Techniques: Labeling, Modification, Control, Cell Culture, Protein Extraction, Magnetic Beads, Data-independent acquisition, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Standard Deviation