Journal: The EMBO Journal
Article Title: Proteolytic profiling of human plasma reveals an immunoactive complement C3 fragment
doi: 10.1038/s44318-025-00598-8
Figure Lengend Snippet: ( A ) Pull-downs with native and denatured (heat-inactivated and carbamidomethylated) C-terminally His-tagged C3-LHF1 were performed on human kidney homogenate. ( B ) Significantly enriched (BH-adj. t-test p -value < 0.05, log 2 FC > 1) proteins in the kidney pull-downs included the IL6 signaling component IL6ST (gp130). ( C ) Thermal proteome profiling by proteome integral solubility alteration assay (TPP-PISA) in human kidney lysates to identify putative C3-LHF1 interacting proteins ( n = 3 technical replicates). Only sign. altered (BH-adj. t-test p -value < 0.05, log 2 FC > 0.58) and plasma membrane-resident proteins are displayed after addition of 1 or 10 µM C3-LHF1 (1 h, 37 °C). Proteins stabilized by interactions with C3-LHF1 display a higher abundance in the treated samples (IL6ST depicted in red). ( D ) C3-LHF1 effect on IL6R/IL6ST signaling was tested in a HEK-Blue IL6Rα/IL6ST reporter line (incubation o/N at 37 °C). C3-LHF1 effect was concentration dependent, and activating, unlike bulk C3 ( n = 4, mean ± SE; two-sided t-test p -value < 0.001 = *; IL6 used at 3 × 10 −6 µg/mL, p -value vs. untreated control: 0.0000001; p -values for 5-10-20 µM C3-LHF1 vs. untreated control: 0.000119, 0.000009, and 0.000006, respectively). ( E ) In HEK-Blue IL6Rα/IL6ST cell line with both IL6 and C3-LHF1 (o/N at 37 °C), a partial competition can be observed at IL6 concentrations of >1 × 10 −4 µg/mL ( n = 4, mean ± SE). ( F ) Tocilizumab (TOC, 2 µg/mL for 3 h at 37 °C) did not inhibit the C3-LHF1 response in the IL6Rα/IL6ST cell line, unlike the IL6 response at 1 × 10 −4 µg/mL ( n = 4, mean ± SE, two-sided t-test p -value < 0.001 = *; p -value IL6 + tocilizumab vs. IL6 at 1 × 10 −4 µg/mL = 1.5 × 10 −9 ). .
Article Snippet: HEK-Blue IL6 reporter line , Invivogen , #hkb-hil6.
Techniques: Solubility, Clinical Proteomics, Membrane, Incubation, Concentration Assay, Control
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