Goat anti rabbit ig hrp

Proteins were separated by SDS–PAGE and transferred to nitrocellulose membranes. In most cases, the protein content was adjusted to 25 μg of total protein per lane using a Nanodrop^® Spectrophotometer ND-1000 to quantify the protein concentration of the samples. Immunoblotting was carried out as previously described^{90 (link)} using a monoclonal antibody against FLAG (1:5000) (Sigma F1804-1MG)) or a polyclonal antibody against 6xHis tag (1:10,000) (Rockland 600-401-382). The secondary antibody Goat anti-Rabbit Ig-HRP (Bio-Rad 172-1019) was added at a 1:20,000 dilution.

Rabbit anti-α1C antibody (1:1000, #AB5156) was from Millipore. Rabbit monoclonal anti-c-fos (1:1000, #2250) and rabbit monoclonal anti-c-jun (1:1000, #9165) antibodies were from Cell Signaling Technology (Danvers, MA, USA), and rabbit anti-NR1 antibody was from Alomone Lab (#AGC-001). Goat antirabbit Ig-HRP (1:5000, #1705046) was from Bio-Rad (Hercules, CA, USA). Bladder samples, oocytes and cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris, 1% v/v NP-40, 0.5% deoxycholic acid, and 0.1% w/v SDS, pH 7.4) containing protease inhibitors (Complete Mini; Roche Applied Science, Indianapolis, IN, USA). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with primary antibody at 4 °C for 1 h after overnight block with 5% nonfat milk. β-actin (Sigma) was used as the internal control. Bands were visualized with Amersham ECL reagent (Arlington Heights, IL, USA). Exposed and developed film was scanned, and the image contrast was corrected with Photoshop (Adobe Systems, San Jose, CA, USA). Images were imported into Adobe Illustrator CS4 (Adobe) for generation of figures. The blots were quantified using FIJI software. Full blots are shown in source data.