data analysis module  (OriginLab corp)

Journal: Communications Biology

Article Title: HuB serves as the central hub for connecting HuR-related mRNAs with translation machinery in inflammation

doi: 10.1038/s42003-025-09228-9

Figure Lengend Snippet: The expression of HuR, HuB, HuC and HuD in various cell lines and mice lungs. HEK293, HeLa, RAW264.7 and U251 cells were treated with TNFα or LPS for 1 h. Subsequently, Western blotting was performed to detect the protein levels of HuR, HuB, HuC and HuD ( A ). Mice lungs were challenged with LPS (1 mg/kg) for 1 h. Then lung homogenates were prepared, Western blotting was performed to detect the protein levels of HuR, HuB, HuC and HuD ( B ). Quantification of the relative levels of HuR and HuB from the western blotting was achieved by normalization against that of β-actin. C Immunofluorescence analysis of the localization and expression of HuB in mice lungs tissue under inflammatory conditions. Mice lungs were challenged with LPS (1 mg/kg) for 1 h, Then frozen sections of mice lungs were prepared. Expression of HuB was detected by HuB antibody. Nuclei were counterstained with DAPI. Scale bar, 50 μm. D The Hel-N1 isoform of HuB is mainly expressed by brain tissues. Total RNA was extracted from various tissues and cells as indicated, and the purified RNA from each sample was transcribed to cDNA, and the HNS domain of HuB was amplified, The PCR products were sent for sequencing, and the information was compared with the NCBI database to identify the isoforms of HuB (Hel-N1 or Nel-N2). Shown is the diagram of the sequences of HuB mRNAs from the different tissues and cells. E Inflammation stimulation enhances the association of HuB with HuR. HEK293 cells were transfected with GFP-HuB plasmid, and then mock-treated or exposed to TNFα (10 ng/mL) for 1 h. The cytosolic lysates were collected for co-IP assay by using GFP antibody, and then subjected to western blotting to detect the interaction of GFP-HuB with HuR. F ITC binding measurements of GST-HuR with GST-HuB. The purified recombinant proteins (GST and GST-HuR) were injected into calorimetric cells before GST-HuB injection. Theoretical titration curves as well as associated KD values were displayed. G , H Time dynamics of HuR shuttles to the cytoplasm in response to inflammatory stimulation. HEK293 cells were challenged with TNFα (10 ng/mL) for different time intervals, the cells were lysed as cytosolic extracts (CE) and nuclear extracts (NE), and the cytoplasmic location of HuR was analyzed by western blotting ( G ) and immunefluorescence staining (H) with HuR antibody. The profiles in the right panel ( H ) show the fluorescence intensity of HuR from line scans in the merged images in the left panel, analyzed by Image Pro Plus software (red line: HuR; blue line: DAPI). Scale bar, 20 μm. I Knockdown of HuB reduces the TNFα-induced cytoplasmic shuttling of HuR. HEK293 cells were transfected with siRNA targeting HuB or a control. After being mock-treated or exposed to TNFα (10 ng/mL) for 1 h, the CE and NE of cells were prepared, the efficacy of HuB knockdown was showed by western blotting with HuB antibody, and the shuttling of HuR was detected by western blotting with HuR antibody. J The ectopic over-expression of HuB but not HuR increases the cytoplasmic location of HuR. Flag-HuR or Flag-HuB together with GFP-HuR were overexpressed in HEK293 cells. The location of GFP-HuR was visualized with a *60 objective on a confocal microscope. Scale bar, 20 μm. The quantification from randomly three independent experiments was shown in the right panel. Quantification shows mean ± SD based on three independent experiments, with significance of the difference determined by one-way ANOVA ( A , B , E , J ). n = 3 animals ( B , C ), n = 3 independent experiments ( A , D – J ), * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance. Source data are available in supplementary data 1 for this figure.

Article Snippet: Curve fitting to a single binding site model was performed using the ITC data analysis module of MicroCal Software provided by the manufacturer.

Techniques: Expressing, Western Blot, Immunofluorescence, Purification, Amplification, Sequencing, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Binding Assay, Recombinant, Injection, Titration, Staining, Fluorescence, Software, Knockdown, Control, Over Expression, Microscopy