P5747

The indicated plant tissues were harvested, weighed, and ground in liquid nitrogen. Total proteins were extracted in protein extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% Glycerol, 5.0 mM DTT, 2.0 mM Na₂MoO₄, 2.5 mM NaF, 1.5 mM Na₃VO₄, 0.5% [v/v] IGEPAL CA-630 [Sigma-Aldrich], 1.0 mM PMSF, 1% [v/v] protease inhibitor cocktail and 1× PhosSTOP phosphatase inhibitor cocktail [Roche]). Plasma membrane proteins were isolated using a Plasma Membrane Protein Isolation Kit (Invent, SM-005-P) according to the manufacturer’s protocol. Protein samples were separated on 4–12% precast SurePAGE gels (GenScript) at 120 V for 1.5 h and transferred onto activated PVDF membranes at 200 mA for 2 h. Immunoblotting was performed using the following antibodies: anti-BZR1 (Youke Biotechnology, Shanghai, China, YKRP082), 1:1000; anti-BRI1 (Agrisera, AS12 1859), 1:5000; anti-SERK3/BAK1 (Agrisera, AS12 1858), 1:5000; anti-FLAG (Sigma, F1804), 1:2500; anti-phosphoserine (Sigma, P5747), 1:500; anti-phosphothreonine (Cell signaling, 9381), 1:500; anti-HA (Covance, MMS-101R), 1:2500; anti-rabbit (MBL, 458), 1:10,000; and anti-mouse (Solarbio, SE131), 1:10,000.

Colonic specimens were dissected to separate the mucosal/submucosal layer from underlying neuromuscular tissues. Samples of colonic muscular tissue or ICSMCs were homogenized in RIPA lysis buffer (Cole Parmer homogenizer, Generalcontrol SpA, Milano, Italy). Homogenates were spun by centrifugation at 20,000 × g for 15 min. at 4°C. Supernatants were then separated from pellets and stored at −80°C. Protein concentration was determined by the Bradford method (Protein Assay Kit; Bio-Rad, Hercules, CA, USA). Equivalent amounts of protein lysates (50 μg for tissues and 10 μg for ICSMCs) were separated by 8% SDS-PAGE for immunoblotting. After transfer onto a PVDF membrane, the blots were blocked and incubated overnight with a rabbit anti-collagen I antibody (Ab34710; Abcam, Cambridge, UK) or a goat anti-transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) antibody (Table1). After repeated washings with TBS-T, appropriate secondary peroxidase-conjugated antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA) were added for 1 hr at room temperature. Immunoreactive bands were then visualized by incubation with chemiluminescent reagents (Immobilon reagent; Millipore, Billerica, MA, USA), and examined by Kodak Image Station 440 for signal detection. To ensure equal sample loading, blots were stripped and reprobed for determination of β-actin by a specific antibody (P5747; Sigma-Aldrich, Milan, Italy).