Western blotting was performed with samples from the cultured medium and the cell lysates (19 (link)). The cell culture media were collected and concentrated using Amicon® Ultra 10K Filter Devices (Millipore). The cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, BP-115). Samples were electrophoresed on 8–14% SDS-PAGE and then transferred to Immobilon-P PVDF transfer membranes (Merck Millipore). The membrane was blocked, probed with primary Abs overnight at 4°C, and then incubated with HRP-conjugated secondary Abs (1:3000~5000) at room temperature for 1 h. The protein bands were detected with Odyssey Fc Imager (LI-COR Biosciences). Relative band densities of proteins were quantified using ImageJ Software (NIH).
Immunoprecipitation was performed as described previously (19 (link)). BMDMs were lysed in immunoprecipitation lysis buffer (1% NP-40, 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) supplemented with 1 mM PMSF and protease inhibitor. The cell lysates were incubated with an appropriate Ab overnight at 4°C, followed by addition of 20 μl protein A/G PLUS-agarose beads for 2 h at 4°C. The resulting immunoprecipitates were dissolved in SDS-PAGE sample buffer for electrophoresis and immunoblot analysis
Immunoprecipitation was performed as described previously (19 (link)). BMDMs were lysed in immunoprecipitation lysis buffer (1% NP-40, 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl) supplemented with 1 mM PMSF and protease inhibitor. The cell lysates were incubated with an appropriate Ab overnight at 4°C, followed by addition of 20 μl protein A/G PLUS-agarose beads for 2 h at 4°C. The resulting immunoprecipitates were dissolved in SDS-PAGE sample buffer for electrophoresis and immunoblot analysis
