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Image Search Results
Journal: bioRxiv
Article Title: The active secretion of a subunit of IL-12 by tissue cells is regulated by Valosin-Containing Protein and intracellular calcium redistribution
doi: 10.64898/2026.01.28.702376
Figure Lengend Snippet: The IL-12p35 subunit is not secreted in the absence of the IL-12p40 binding partner: Development of two novel reporter systems to track the release of p35 from cells and subcellular location throughout p40-independent release. (A) Luciferase assay to detect p35NL release in the cell culture supernatant when co-expressed with p40. HEK cells were transfected with p35NL + empty vector (light red), p35NL + p40 (red), NanoLuc-only (NLO) + empty vector (light blue), or NLO + p40 (blue). Supernatants were collected 48 hours after transfection for luciferase assay (n=3). Luminescence data is represented in Raw Luminescence Units (RLUs). Functionality of p35NL as an IL-12 subunit when co-expressed with p40. SMARTA transgenic T cells were activated in vitro and re-stimulated with either recombinant p40 protein, recombinant IL-12, or supernatants from HEK cells co-transfected with p35NL and p40. After 2 hours of re-stimulation, IFN-γ production by T cells was measured using intracellular cytokine staining. (B) MFI of live CD4 + CD44 + IFN-γ + SMARTA transgenic T cells populations after restimulation (n = 3). (C) Percentages of MFI of live SMARTA CD4 + CD44 + IFN-γ + transgenic T cells populations after restimulation (n = 3). (D) Representative flow plots compare the percentage of live CD4+CD44+IFNγ+ cells among groups (n = 3). Characterization of fluorescent reporter system for tracking the intracellular location of p35 through ER and Golgi compartments. (E) Murine fibroblasts (L cells) were retrovirally transduced with p35-Scarlet, Golgi-eGFP (Beta-1,4-galactosyl transferase 1), and ER-Halotag (KDEL tagged with Halotag and developed with JFX650). Images are representative of steady-state cells during live cell fluorescent imaging at 100X. White scale bars correspond to 10µm. Characterization of fluorescent reporter system for tracking the association of p35 with ER chaperone, BiP. (F) L cells were retrovirally transduced with p35-Scarlet, Golgi-eGFP, and BiP-BFP. Images are representative of steady-state cells during live cell fluorescent imaging at 100X. White scale bars correspond to 10µm. (G) Pixel intensity of each marker in p35-Scarlet/Golgi-GFP/ER-Halotag L cells along the ROI line draw in ‘MERGED (F)’. ROI line is designated by an arrow. (H) Pixel intensity of each marker in p35-Scar/Golgi-GFP/BiP-BFP L cells along the ROI line draw in ‘MERGED (G)’. ROI line is designated by an arrow. Data is presented as the mean with SEM error bars. Significant changes in luminescence in (A) were determined using an unpaired t-test. Significant changes in percentage and MFI in (B) and (C) were determined using an unpaired t-test. p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant.
Article Snippet: The p35-Scarlet (p35-Scar) construct was generated by amplifying murine p35 cDNA from a plasmid containing the
Techniques: Binding Assay, Luciferase, Cell Culture, Transfection, Plasmid Preparation, Transgenic Assay, In Vitro, Recombinant, Staining, Transduction, Imaging, Marker