Amicon ultra 30 000

The conditioned serum-free medium from THP-1 cells with or without exposure to IFN-α (50 ng/ml), IFN-β (10 ng/ml) or LPS (0.1 μg/ml) for 24h, by CpG ODN (1 μM) or poly(I:C) (10 μg/ml) for 12h was concentrated (40-fold) using Amicon Ultra 30,000 (Millipore). The lysoPLD activity in the concentrated conditioned medium was analyzed using fluorogenic substrate FS-3 as described previously [33 (link)]. Briefly, the assays were performed by mixing 50 μl concentrated medium with 10 μM FS-3 at 37°C for 4 h. LysoPLD activity was measured by detecting the fluorescence increase with 494 and 520 nm as the excitation and emission wavelengths, respectively.

Cells were lysed in RIPA buffer for 30 min. After centrifugation, the supernatants were quantified by bicinchoninic acid assay (Micro BCA; Pierce Biotechnology, Rockford, IL). For experiments detecting the secreted ATX protein, the culture medium was concentrated (by approximately 30-fold) using Amicon Ultra 30,000 (Millipore). Protein quantification was conducted and equal amount protein was loaded for each sample. Protein samples were subjected to SDS-PAGE and analyzed as described previously [33 (link)]. Each Western blot analysis was repeated at least three times.

The cells were lysed in RIPA buffer (Macgene, Beijing, China, #MP015) supplemented with a protease inhibitor (MedChemExpress, Monmouth Junction, NJ, USA, HY-K0012). After determining the protein concentration using a BCA Protein Assay Kit (Thermo Fisher Scientific, Bremen, Germany, #23227), equivalent protein quantities were subjected to SDS–PAGE and then transferred to PVDF membranes (Millipore, Darmstadt, Germany, #GVWP02500). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then probed with the indicated primary antibodies, followed by the appropriate HRP-conjugated anti-mouse/rabbit secondary antibodies (Zsgb, Beijing, China, #ZB-2301). Immunoreactive bands were measured with an enhanced chemiluminescence western blotting system (Millipore, Darmstadt, Germany, #WBKLS0500). For the detection of ATX, a secreted protein, cells were cultured in serum-free medium. The medium was concentrated (20-fold) using an Amicon Ultra 30000 (Merck KGaA, Darmstadt, Germany), and then subjected to SDS-PAGE and western blotting.