Dc protein assay

Total protein was isolated from approximately 50 mg of snap-frozen colon using Cell Extraction Buffer (Invitrogen, Grand Island, NY) containing Halt protease and phosphatase inhibitors (ThermoFisher Scientific, Waltham, MA) and Na₃VO₄ (Sigma, St. Louis, MO). Protein concentrations were determined using a D_C protein assay (ThermoFisher). Samples were reduced by heating to 95°C for 5 minutes in the presence of 2-mercaptoethanol, subjected to SDS-PAGE (Criterion 4% to 12% Bis-Tris gels; Bio-Rad, Hercules, CA), and transferred to Nitrocellulose transfer membrane (Whatman GmbH, Dassel, Germany) by Criterion Blotter wet transfer (Bio-Rad). The membranes were blocked for 1 hour at room temperature in 5% milk in Tris-buffered saline with Tween-20 (TBST) and incubated overnight at 4°C with antisera to CRF₁ (1:250; Millipore, Billerica, MA), CRF₂ (1:1000; Millipore), and GAPDH (1:2000; Cell Signaling Technology, Danvers, MA) diluted in 5% milk in TBST. Membranes were then washed with TBST and incubated for 1 hour with anti-rabbit secondary antibody (1:2000; Cell Signaling, Danvers, MA). Densitometry was performed using Quantity One 4.6.9 software (Bio-Rad).

Total protein was isolated from approximately 50 mg of snap-frozen vagina, bladder, and colon using Cell Extraction Buffer (Invitrogen, Grand Island, NY) containing Halt protease and phosphatase inhibitors (ThermoFisher Scientific, Waltham, MA) and Na₃VO₄ (Sigma, St. Louis, MO). Protein concentrations were determined using a D_C protein assay (ThermoFisher). Samples were reduced by heating to 95°C for 5 minutes in the presence of 2-mercaptoethanol, subjected to SDS-PAGE (Criterion 4% to 12% Bis-Tris gels; Bio-Rad, Hercules, CA), and transferred to Nitrocellulose transfer membrane (Whatman GmbH, Dassel, Germany) by Criterion Blotter wet transfer (Bio- Rad). The membranes were blocked for 1 hour at room temperature in 5% milk in Tris-buffered saline with Tween-20 (TBST) and incubated overnight at 4°C with antisera to CRF₁ (1:500; Millipore, Billerica, MA), CRF₂ (1:800; Millipore), TRPV1 (1:1000; Alomone Labs, Jerusalem, Israel), or TRPA1 (1:1000; Aviva Systems Biology, San Diego, CA) and GAPDH (1:2000; Cell Signaling Technology, Danvers, MA) diluted in 5% milk in TBST. Membranes were then washed with TBST and incubated for 1 hour with antirabbit secondary antibody (1:10,000; Cell Signaling, Danvers, MA). Densitometry was performed using Quantity One 4.6.9 software (Bio-Rad).

Total proteins were isolated from approximately 50 mg of snap-frozen DRG, vagina, and colon tissue samples using Cell Extraction Buffer (Invitrogen, Grand Island, NY) containing Halt protease and phosphatase inhibitors (ThermoFisher Scientific, Waltham, MA) and Na₃VO₄ (Sigma, St. Louis, MO). Protein concentrations were determined using a D_C protein assay (ThermoFisher). Samples were reduced by heating to 95°C for 5 minutes in the presence of 2-mercaptoethanol, subjected to SDS-PAGE (Criterion 4% to 12% Bis-Tris gels; Bio-Rad, Hercules, CA), and transferred to Nitrocellulose transfer membrane (Whatman GmbH, Dassel, Germany) by Criterion Blotter wet transfer (Bio-Rad). The membranes were blocked for 1 hour at room temperature in 5% milk in Tris-buffered saline with Tween-20 (TBST) and incubated overnight at 4°C with antisera to CRF₁ (1:500; Millipore, Billerica, MA), TRPV1 (1:1000; Alomone Labs, Jerusalem, Israel), or TRPA1 (1:1000; Aviva Systems Biology, San Diego, CA) and GAPDH (1:2000; Cell Signaling Technology, Danvers, MA) diluted in 5% milk in TBST. Membranes were then washed with TBST and incubated for 1 hour with anti-rabbit secondary antibody (1:10,000; Cell Signaling, Danvers, MA). Densitometry was performed using Quantity One 4.6.9 software (Bio-Rad).