Anti trpa1 antibody

An anti-TRPA1 antibody (1:500 dilution) was obtained from Novus Biologicals (Littleton, CO, USA). The selective TRPA1 antagonists HC-030031 (HC) and TCS-5861528 (TCS) were purchased from R&D Systems (Minneapolis, MN, USA). Wheat germ agglutinin (WGA, 1:200 dilution) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for T-CaMKII (1:1000 dilution), calcineurin (1:1000 dilution), GAPDH (1:2500 dilution), α-smooth muscle actin (α-SMA, 1:500 dilution), CD3 (1:200 dilution), and CD68 (1:200 dilution) were purchased from Abcam (Cambridge, MA, USA). An antibody for p-CaMKII (1:1000 dilution) was purchased from GeneTex (Irvine, CA, USA). Antibodies for CD206 (1:200 dilution) were purchased from R&D Systems (Minneapolis, MN, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD206, allophycocyanin (APC)-conjugated anti-F4/80, phycoerythrin (PE)-conjugated anti-CD45 and FITC-conjugated anti-CD3 were purchased from BioLegend (San Diego, CA, USA). Secondary antibodies and goat anti-rabbit IgG were obtained from LI-COR Biosciences (Lincoln, NE, USA). All other chemicals were of analytical grade.

Tissue samples (~1 g) of the distal colon and the spinal cord were extracted in three volumes of extract buffer, centrifuged at 13,000× g for 10 minutes at 4°C; the supernatant was saved at −70°C until usage. Western blotting protocol was developed for the Bio-Rad protein gel and transfer apparatus. The membranes were blocked and incubated with the primary antibodies at 4°C, overnight, and subsequently with the secondary antibodies for 2 hours. The gel was transferred to a polyvinylidene difluoriizde membrane and blotted with primary (overnight) and then with secondary (1 hour) antibodies. The primary antibodies included a rabbit polyclonal anti-TRPA1 antibody (1:200; Novus Biologicals) and a mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:500; Sigma). The secondary antibodies included anti-mouse Ig for GAPDH (1:1,000) and anti-rabbit Ig for TRPA1 (1:3,000). The integrated density of Western blotting bands was quantitatively analyzed by the Gel Image analysis software. TRPA1 protein expression was normalized to GAPDH.

IOSE80 human ovarian surface epithelial cell line (Guandao Biological Engineering, China), A2780 human ovarian carcinoma cell line (KeyGen Biotech, China), and its taxol-resistant strain were cultured in RPMI 1640 medium. HepG2 human hepatocellular carcinoma cell line (ATCC) was cultured in MEM medium. SK-OV-3 human ovarian adenocarcinoma cell line (ATCC, USA), HFTEC fallopian tube cell line, and MCF-7 human breast cancer cell line (ATCC) were cultured in DMEM. All the cell lines were cultured in medium that contained 10% FBS, 1% penicillin/streptomycin (P/S) in a humidified 5% CO₂ atmosphere at 37°C. For western blot assay, primary antibodies were purchased from the indicated sources, anti-TRPA1 antibody (1:500 dilution, Novus, USA); anti-β-actin antibody (1:1,000 dilution, Santa Cruz, USA). A fluorescent secondary antibody against rabbit was purchased from Li-cor (USA). For immunoprecipitation assay, anti-AGO2 antibody was purchased from Active Motif (USA), and anti-rat IgG antibody was purchased from Santa Cruz. For immunohistochemistry assay, primary antibodies were purchased from the indicated sources, anti-ki67 antibody (1:1,000 dilution, Abcam, USA) or anti-PCNA antibody (1:1,000 dilution, Novus, USA). A GTVison secondary antibody (GK600710A, HRP-conjugated anti-rabbit/mice antibody) was purchased from Gene Tech (Shanghai, China).