1 2 diamino 4 5 methylenedioxybenzene

Samples of ear, tail and small intestine weighing approximately 100 mg were cut into small pieces in MQ water and incubated at 95°C for 30 min. After the samples had cooled to room temperature, 0.5 M H₂SO₄ was added to a final concentration of 25 mM. The mixtures were incubated at 80°C for 1 h to release the sialic acids from the samples. After centrifugation, the supernatant was collected, an equal volume of DMB (1,2-diamino-4,5-methylenedioxybenzene, Sigma-Aldrich, Inc.) solution (1.6 mg DMB in 1 mL of 1.4 M acetic acid, 0.75 M 2-mecaptoethanol and 18 mM sodium hydrosulfite solution) was added, and the mixture was incubated at 80°C for 2 h to label the sialic acids. The labelled NGNA and NANA used as standards were prepared as 1 mg/mL solutions and reacted under the same labelling conditions. The DMB-labelled sample was injected onto a Waters^™ HPLC system (Waters 2475 Multi-wavelength Fluorescence Detector, Waters 717 plus Autosampler and Waters 600 Controller) with the Discovery BIO wide Pore C18 (5 μm, 4.6 x 25 cm) column. The analysis was performed using an isocratic mobile phase of methanol:acetonitrile:H₂O (7:9:84) at a flow rate of 0.6 mL/min; the fluorescence detector was set at an excitation wavelength of 373 nm and an emission wavelength of 448 nm. (dx.doi.org/10.17504/protocols.io.zd6f29e).

Neu5Gc was purchased from Aladdin (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), trypsin, fetal bovine serum, penicillin, streptomycin, phosphate-buffered saline (PBS), Hank’s Balanced Salt Solution (HBSS) were purchased from BI (Kibbutz Beit-Haemek, Israel). Mouse anti-ZO-1 antibody Alexa Flour 488 conjugate were purchased from Invitrogen (San Francisco, CA, USA). Anti-ZO-1 and occludin antibodies were purchased from Affinity (Changzhou, China). Anti-claudin-1, p65, p-p65, I-κBα, p-I-κBα, and anti-β-actin antibodies were purchased form Abmart (Shanghai, China). BAY 11-7082 was purchased from Beyotime (Shanghai, China). Fish skin gelatin, 1,2-diamino-4,5-methylene-dioxybenzene (DMB), and sodium azide was purchased from Sigma (St. Louis, MI, USA). Chlorpromazine hydrochloride, amiloride hydrochloride and the MTT Detection Kit were purchased from Solarbio (Beijing, China). Dynasore was purchased from MCE (Monmouth Junction, NJ, USA). Nocodazole and nystatin were purchased from Yuanye Bio-Tech (Shanghai, China). Monensin sodium salt and cytochalasin D were purchased from Aladdin (Shanghai, China). Bafilomycin A1 was purchased from LC Laboratories (Woburn, MA, USA). Brefeldin A was purchased from Selleckchem (Houston, TX, USA).