Fig 2: CK2 phosphorylates RIOK1 at T410 both in vitro and in vivo.(A) RIOK1 consensus phosphorylation sites (shown in red) corresponding to the CK2 consensus motifs S/TXXE/D are presented. These consensus phosphorylation sites are exactly located within the consensus sequences of SETD7. (B) FLAG-RIOK1 was present in the HA-CK2 immunocomplex. Total cell lysates were extracted from 293T cells transiently co-transfected with FLAG-RIOK1 and HA-Vec and HA-CK2α subjected to immunoprecipitation with an anti-HA antibody followed by immunoblotting with indicated antibodies. (C) In vitro glutathione S-transferase (GST)-precipitation assay of no CK2α, or purified His-tagged CK2α combined with GST alone or GST-RIOK1. (D) Immunoprecipitation and immunoblot analysis of lysates of HEK293T cells expressing no plasmid (−) or plasmid encoding the Flag-tagged amino terminus (amino acids 1–242) of RIOK1 (Flag–RIOK1-N) or carboxyl terminus (amino acids 243–568) of RIOK1 (Flag–RIOK1-C), plus Myc-tagged CK2α, probed with anti-Myc and/or anti-Flag. (E) Lysates of HEK293T cells expressing Flag-tagged wild-type RIOK1 and Myc-tagged wild-type CK2α or CK2α-K68A; far right (λ-PPase), was analyzed using Western blot. GAPDH serves as a loading control throughout. (F) Immunoblot analysis of RIOK1 and CK2α in lysates of HEK293T cells expressing Flag-tagged wild-type RIOK1 or RIOK1 (T410A) and Myc-tagged wild-type CK2α or CK2α-K68A. (G) Immunoblot analysis of phosphorylated p-RIOK1-T410 and total RIOK1 and CK2 in lysates of HEK293T cells. (H) In vitro kinase assay of purified recombinant GST-tagged RIOK1 or RIOK1-T410A with HA-tagged CK2.

Fig 3: IL-31RA augments CHRM3-driven calcium signaling and MLC phosphorylation in ASMC.(A) Quantification of the transcripts of different isoforms of CHRMs in the lungs of wild-type and IL-31RA-/- mice. (B) Quantification of the transcripts of CHRM3 in ASMC isolated from wild-type mice and treated with media, IL-4 (10 ng/ml) or IL-31 (500 ng/ml) for 16 h. (C) Quantification of the transcripts of CHRM3 in ASMC isolated from IL-31RA-/- mice and treated with media, IL-4 (10 ng/ml) and IL-31 (500 ng/ml) for 16 h. (D) ASMC isolated from wild-type and IL-31RA-/- mice were lysed and immunoblotted with antibodies against CHRM3 and GAPDH. CHRM3 protein levels were normalized to GAPDH and shown as fold induced using a bar graph (***P < 0.0005; n = 3, Student’s 2-tailed t test). (E) HEK293T cells were transiently co-transfected with overexpressing plasmids for CHRM3 and IL-31RA or empty control plasmids for 48 h. The IL-31RA-CHRM3 complex formation was visualized using hybridization probes labeled with Alexa 594 (Red). The nuclei were stained with DAPI (blue) and images were captured at 40X magnification. Scale bar, 100 µm. (F) Western blot analyses of CHRM3 in anti-FLAG or control IgG immunoprecipitants from cell lysates of HEK293T cells transiently transfected with a control plasmid or IL-31RAOE plasmid for 72 hrs. Total cell lysates and eluted fractions were immunoblotted with anti-Flag, anti-IL31RA and anti-CHRM3 antibodies. The data presented is a representative of three independent experiments with similar results. (G) Increases in carbachol-induced intracellular calcium levels were measured in HEK293T cells transiently transfected with overexpressing plasmids for CHRM3 and IL-31RA or empty control plasmids for 72 h and treated with carbachol (10µM). (H) HEK293T cells were transiently co-transfected with overexpressing plasmids for CHRM3 and IL-31RA or empty control plasmids for 48 h and treated with carbachol (10µM) for 10 min. Cell lysates were immunoblotted with antibodies against phospho-MLC, total-MLC and GAPDH. Data are shown as means ± SEM, n = 3/group. (I) ASMC isolated from wild-type and IL-31RA-/- mice were treated with carbachol (10µM) for 10 min and cell lysates were immunoblotted with antibodies against phospho-MLC, total-MLC and GAPDH. Data are shown as means ± SEM, n = 3/group