Criterion 4 12 bis tris gels

Mice were overdosed with inhaled isoflurane (<5%) and dura was removed and flash frozen in liquid nitrogen before storage at −80°C. Dural protein was isolated using Cell Extraction Buffer containing Halt protease and phosphatase inhibitors (Thermo-Fisher Scientific, Waltham, MA, United States) and Na₃VO₄. Protein concentrations were determined using a Pierce BCA assay (Thermo-Fisher Scientific, Waltham, MA, United States). Samples were reduced by heating to 95°C in the presence of 2-mercaptoethanol, subjected to SDS-PAGE (Criterion 4–12% Bis-Tris gels; Bio-rad laboratories), and transferred to Nitrocellulose transfer membrane (Whatman GmbH, Dassel, Germany) by Criterion Blotter wet transfer (Bio-Rad). The membranes were blocked for 1 h in 5% milk in Tris-buffered saline with Tween-20 (TBST) then incubated overnight with protease-activated receptor 2 (PAR2; 1:1000; Abcam), transient receptor potential ankyrin 1 (TRPA1;1:1000; Aviva) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH;1:2000; Cell Signaling) antisera. Membranes were washed with TBST and incubated for 1 h with anti-rabbit secondary antibody (1:10,000; Cell Signaling). Densitometry was performed using Quantity One 4.6.9 software (Bio-Rad Laboratories). PAR2 and TRPA1 protein levels were normalized to GAPDH.