Fig. 2: nex-IL1β-mRNA binds to ZC3H12D on the cell surface.

a Representative images showing the colocalization of IL1β-mRNA-FITC and ZC3H12D in ZC3H12D-overexpressing RAW cells (ZC⁺RAW) 30 min after TCM stimulation. The image after stimulation by NoCM as the control is shown in Supplementary Fig. S2a. Merged data with actin filament marker, phalloidin, and DAPI were shown. Bars, 5 μm. Experiments were repeated twice with similar results. b Western blot analysis of RAW and ZC⁺RAW. Anti-ZC3H12D and anti-FLAG antibodies detected FLAG-tagged (C-term) ZC3H12D, and immunoprecipitated ZC3H12D after TCM application plus UV-crosslink is shown (top). The binding of IL1β-mRNA in lung TCM (bottom) to ZC3H12D-FLAG was detected by qPCR analyses. The averages of two independent experiments were shown in the graphs. Repeated experiment data related to IL1β-RNA are shown in Supplementary Fig. S9. c, e Autocorrelation curves obtained by FCS measurements of the labeled ZC3H12D protein in the absence and presence of non-labeled RNA. Alexa Fluor 488-labeled ZC3H12D protein (1 nM, dotted line) was mixed with various concentrations of mouse IL1β-RNA (10 nM, solid (red) line) or βactin-RNA (10 nM, solid (blue) line) (c), and IL1β(3′-UTR)-RNA (5 nM, solid (magenta) line) or IL1β(3′-UTR)-RNA (10 nM, solid (purple) line) (e). d, f Fractions of the ZC3H12D-RNA complex relative to the total content of ZC3H12D obtained by a three-component analysis of the autocorrelation data. Source data are provided as a Source Data file.