Radioimmunoprecipitation assay reagent

Following the manufacturer's instructions, total proteins were extracted using radioimmunoprecipitation assay reagent (Beyotime Institute of Biotechnology, Haimen, China), the bicinchoninic acid assay (Beyotime Institute of Biotechnology) was used to determine concentration of the protein lysate. Following denaturation in loading buffer, the protein lysate was separated by 15% SDS-PAGE gel and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h and then incubated with the primary antibody against TRPV1 (1:2,000), SP (1:500), CGRP (1:500) and β-actin (1:4,000; ab8229; Abcam) overnight at 4°C. Following washing in TBS containing 1% Tween-20 (TBST), the membranes were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000; sc2357; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Following three washes with TBST, the blots were visualized using an electrochemiluminescence system (Image Quant LAS 4000 mini analyzer; General Electric, Fairfield, CT, USA) with Image Quant LAS 4000 mini control software (version 1.2; General Electric). The relative band intensities were analyzed using Image Lab software (version 4.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The experiment was repeated three times.

Total protein was extracted from pericytes using radio immunoprecipitation assay reagent (Beyotime Biotechnology, Shanghai, China) containing 100 μg/mL phenylmethylsulfonyl fluoride (Solarbio) and quantified using a bicinchoninic acid protein assay kit (Solarbio). Protein samples were denatured, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride membrane (EMD Millipore Corp., Burlington, MA, USA). The membranes were then blocked with a blocking solution (QuickBlock, Beyotime Biotechnology) at room temperature for 30 minutes, and then incubated with primary antibody against pro-caspase-1 (1:1000, rabbit, Cat# ab179515, Abcam), Nod1 (1:500, mouse, Cat#sc-398696, Santa Cruz), IL-1β (1:500, rabbit, Cat# ab9722, Abcam), CD9 (1:2000, rabbit, Cat# ab92726, Abcam), CD81 (1:5000, rabbit, Cat# ab109201, Abcam), CD63 (1:500, mouse, Cat#sc-5275, Santa Cruz) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; mouse, 1:1,0000, Cat# AC033, ABclonal, Wuhan, China) at 4°C overnight. The membranes were incubated with secondary goat anti-rabbit or mouse horseradish peroxidase-linked antibody (1:1000, Abbkine, Cat# A21220 or Cat# A25012) at room temperature for 1 hour. Finally, target bands were visualized using an enhanced chemiluminescence western blot kit (Thermo Fisher Scientific) and semi-quantified using ImageJ software.