Ab126168

Western blot (WB) assays were done with rabbit anti-human CHRM3 (cat Ab126168, Abcam, Cambridge, UK) and mouse anti-actin antibodies (cat mAbcam 8226, Abcam, Cambridge, UK). In brief, the samples were lysed with RIPA buffer (cat R0010, Solarbio, Beijing, China) containing protease inhibitors (SKU 11836153001, Roche, Basel, Switzerland). The lysates were divided on 10% SDS-PAGE gels (cat 20325ES62, Yeason, Shanghai, China). The membranes (cat GVWP02500, Millipore, MA, USA) were incubated with rabbit anti-human CHRM3 antibody (1:1000) or mouse anti-actin antibody (1:10,000). HRP-conjugated secondary antibodies were used, and bands were spotted using an ECL kit (cat PI32209, Thermo Scientific Pierce, Waltham, MA, USA).

Cells were first fixed with 4% paraformaldehyde for 20 min at room temperature. Following the removal of the fixative by washing with PBS, cells were treated with cold methanol for 20 min at −20 °C for permeabilization. Five percent FBS (Thermo Fisher Scientific, Waltham, MA, USA) in PBS was applied for 2 h to block unspecific binding. Primary antibodies for MIST1 (Abcam, ab107390,1:100), rabbit polyclonal anti-TCF3 antibody (Santa Cruz, sc763, 1:100), goat polyclonal anti-cMyc antibody (Bethyl, A190-104A, 1:100), rabbit polyclonal anti-AMY1 antibody (Biomatik, CAU25843, 1:100), anti-rabbit polyclonal anti-M3R antibody (Abcam, ab126168, 1:100), rabbit polyclonal anti-AQP5 (Abcam, Ab78486, 1:100), and anti-rabbit polyclonal anti-CK19 antibody (Abcam, ab52625, 1:100) were diluted in 5% FBS blocking buffer and applied on cells overnight at 4 °C. An anti-rabbit/goat Alexa Fluor™ 568 or 488 nm conjugated antibodies (1:200) was used as a secondary antibody for 2 h at room temperature followed by a final wash. Endoplasmic reticulum staining was performed by an ER-GFP, BacMam 2.0 labeling kit (Cell light, C10590). Images were obtained using a Zeiss Axiovert 200M microscope equipped with a AxioCam MRm camera and AxioVs40 software (Ver. 4.7.1.0) (Zeiss, Oberkochen, Germany).

HEK293T cells were transiently transfected with overexpressing plasmids for IL-31RA (Origene, RC218212L1) and CHRM3 (Origene, SC119736) or control empty plasmids (pLJM1-EGFP, Addgene 19319 and pLenti-C-Myc-DDK-P2A-Puro, Origene PS100092) using Lipofectamine 3000 and cultured for 48 h. Then, cells were fixed for 5 min in ice cold methanol at 4°C and incubated with Duolink blocking solution (Sigma) for 1 hour at room temperature followed by overnight incubation with primary antibodies for IL-31RA (15 μg/ml, R&D Systems AF2769) and CHRM3(1:500, Abcam ab126168). The incubation was followed by three 5 min washes and incubation with PLA probes anti-goat PLUS (DUO92003) and anti-rabbit MINUS (DUO92005) for 1 h at 37°C as recommended in the Duolink PLA kit (Sigma). After three washes and cells were incubated with the hybridization solution containing DNA ligase for 30 min at 37°C. Cells were washed and incubated with the amplification-polymerase at 37°C for 100 min, washed three times and mounted with DAPI mounting medium. To identify the membrane localization of in situ PLA signals, the cells were counterstained with cholera toxin conjugated with Alexa Fluor 488 (1:200, Thermo Fischer). Fluorescence images were acquired using a Nikon A1R confocal laser scanning microscope and analyzed using Imaris image analysis software.