Ab134306

Tissues and cells were lysed by RIPA lysis buffer (P0013b, Beyotime, China). Proteins were separated using SDS-PAGE and blotted onto PVDF membranes (Millipore, USA). The blots were then blocked with 5% non-fat milk and incubated with specific primary antibodies against CD9 (ab307085, Abcam, USA), CD63 (ab134045, Abcam, USA), CD81 (ab79559, Abcam, USA), TSG101 (ab125011, Abcam, USA), Calnexin (ab133615, Abcam, USA), Bcl-2 (ab32124, Abcam, USA), Bax (ab32503, Abcam, USA), cleaved Caspase-3 (ab2303, Abcam, USA), Caspase-3 (ab32351, Abcam, USA), GFAP (ab7260, Abcam, USA), NF200 (ab134306, Abcam, USA), TLR4 (ab13556, Abcam, USA), p65 (ab32536, Abcam, USA), IκB (9242, CST, USA), pIκB (9246, CST, USA), H3 (ab1791, Abcam, USA), β-actin (ab8226, Abcam, USA) overnight at 4 °C. Next day, the blots were probed with HRP-conjugated anti-rabbit (ab205718, Abcam, USA) or anti-mouse (ab205719, Abcam, USA) secondary antibodies at room temperature for 1 h. After reaction with ECL reagent (Millipore, USA), the protein bands were visualized by a gel image system.

Schwann cells and sciatic nerve were lysed with RIPA lysis buffer (Beyotime, Shanghai, China), and the quantity of the protein in the lysate was quantified using the BCA protein quantification kit (Beyotime Biotechnology, Shanghai, China). After loading buffer was added, the extracted protein was denatured in boiling water, and then the protein samples were separated by 10% SDS-PAGE and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). Then, these membranes were blocked with 5% skim milk at room temperature for 30 min, and primary antibodies, including anti-NGF (Abcam; 1:1000; ab6199), anti-myelin-associated glycoprotein (MAG) (Abcam; 1:1000; ab89780), anti-peripheral myelin protein-22 (PMP22) (Abcam; 1:1000; ab270400), anti-growth-associated protein-43 (GAP43) (Abcam; 1:1000; ab16053), anti-neurofilament 200 (NF200) (Abcam; 1:1000; ab134306), and anti-β-actin (Abcam 1:3000; ab8227) were added to incubate the membranes at 4°C overnight. The next day, after being washed twice with TBST, the membranes were incubated with the HRP-conjugated secondary antibody for 1 h at room temperature. The membrane was rinsed for 3 times before the protein bands were visualized with enhanced chemiluminescence solution (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) exposed on film.

Trypsin-3 (10 nM) was added to the submucosal tissue that was isolated from colonic biopsies and cultured in neurobasal medium supplemented with 10% heat-inactivated FBS, 1% antibiotic-antimycotic solution for 2 hours. The plexus was then washed in phosphate buffer saline (PBS), fixed in paraformaldehyde 4% and processed for immunostaining, using chicken antineurofilament 200 kDa (NF200, 1:500, ab134306-Abcam), to identify neurons and nerve fibres, and mouse anti-PAR₂ antibody (1:200, SAM-11-LifeSpan BioSciences, Seattle, USA).13 (link) Primary antibody incubation (overnight at 4°C) was followed by incubation with appropriate fluorescently labelled secondary antibodies (2 hours room temperature). The tissue was mounted on a microscope slide in Citifluor (Citifluor, Leicester, UK). PAR₂ involvement specificity was investigated in the presence of the PAR₂ antagonist GB83 (10 μM),14 (link) added 30 min before trypsin-3.