Fig. 9. The post-transcriptional regulation of CHRM3 expression by IL-31RA.

a Quantification of CHRMs transcripts in the lungs of wildtype (n = 5) and IL-31RA^–/– (n = 5) mice using RT-PCR. Data shown as mean $\pm$SEM. Two-way ANOVA was used. b, c Quantification of CHRM3 transcripts in ASMC isolated from wildtype (n = 3) and IL-31RA^–/– (n = 3) mice and treated with media, IL-4 (10 ng/mL) or IL-31 (500 ng/mL) for 16 h. Data shown as mean $\pm$SEM. Two-way ANOVA was used. d, e The total lung lysates of HDM-treated wildtype (n = 5) and IL-31RA^–/– (n = 5) mice were immunoblotted with antibodies against CHRM3 and GAPDH. Data are shown as mean ± SEM. Unpaired t test was used. f ASMC isolated from wildtype (n = 3) and IL-31RA^–/– (n = 3) mice were lysed and immunoblotted with antibodies against CHRM3 and GAPDH. Data are shown as mean $\pm$SEM. Unpaired t test was used. g hTERT cells were transiently transfected with IL-31RA-specific (n = 3) or control siRNA (n = 3) for 72 h. Cell lysates were immunoblotted with antibodies against IL-31RA, CHRM3 and β-actin. Bar graph shows CHRM3 protein levels normalized to β-actin. Unpaired t test was used. Data are shown as means ± SEM. h Cell surface proteins were biotinylated, and affinity purified using neutravidin to measure cell surface levels of IL-31RA and CHRM3 in hTERT cells transfected with IL31RA-specific (n = 2) or control siRNA (n = 2) for 72 h. hTERT cells without biotinylation were used as a negative control. CHRM3 protein levels were normalized to ITGB1. i HEK293 cell were transiently transfected with overexpressing plasmids for CHRM3 and IL-31RA or empty control plasmids for 48 h. Cells were treated with antibodies against IL-31RA and CHRM3 and the IL-31RA-CHRM3 complex formation was visualized using hybridization probes at an excitation λex 594 nm (Red). The plasma membrane was stained with cholera toxin subunit b conjugated with Alexa Fluor 488 (Green) and the nuclei with DAPI (Blue). The white arrowheads highlight colocalization between the cholera toxin and puncta of the IL-31RA-CHRM3 complex. Images were captured at ×40 magnification. Scale bar, 50 µm. j hTERT cells were treated with antibodies against IL-31RA and CHRM3 or isotype IgG (negative PLA staining). The IL-31RA-CHRM3 complex formation was visualized as described in Fig. 9i. k HEK293T cells transiently transfected with a control plasmid or FLAG-tagged IL-31RA^OE plasmid for 72 h. Total cell lysates and eluted fractions were immunoblotted with anti-Flag, anti-IL31RA and anti-CHRM3 antibodies. At least two independent experiments produced similar results. Source data are provided as a Source Data file.