Tumor necrosis factor alpha tnfα

An ice-cold RIPA buffer (CST) supplemented with a protease inhibitor cocktail (ethylenediaminetetraacetic acid-free) and a phosphatase inhibitor cocktail (Roche Diagnostics Ltd.) was used for homogenization of RWPE-1 cells and rat prostate tissue. After homogenization, samples were cooled to 4 and centrifuged at 15,000 g for 15 minutes to separate the supernatant, and the proteins were transferred to a nitrocellulose membrane. After membrane transfer, the membrane was blocked for 1 hour at room temperature and incubated with primary antibodies to help minimize potential for content overlap. The primary antibodies used were cannabinoid receptor 2 (CB2) (dilution 1:500; Abcam), transient receptor potential vanilloid 1 (TRPV1) (dilution 1:1000; Abcam), tumor necrosis factor-alpha (TNF-α) (dilution 1:1000; Santa Cruz), nuclear factor-kappa B (NF-κB) (dilution 1:500; CST), cyclooxygenase-2 (COX2) (dilution 1:500; CST), interleukin-6 (IL-6) (dilution 1:1000; CST), toll-like receptor 4 (TLR4) (dilution 1:1000; CST), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (dilution 1:5,000; Abcam) followed by membrane-binding to horseradish peroxidasebound secondary antibodies and incubation for 1 hour at room temperature. Enhanced chemiluminescence (Amersham) was used to detect proteins. Images were analyzed using ImageJ (NIH) to quantify the individual protein bands.