Anti syk antibody

Western blotting analysis was performed as described with slight modifications (Tanaka et al., 2014 (link)). Peritoneal macrophages were stimulated with Mincle ligands for 24 h and lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 7.4) supplemented with Protease Inhibitor Cocktail (Merck) and Phosphatase Inhibitor Cocktail 2 and 3. Proteins were separated by SDS-PAGE and immunoblotted with anti-phospho Syk (Tyr525/526) antibody (diluted 1:500; #2710; Cell Signaling Technology) and anti-Syk antibody (diluted 1:1,000; #13198; Cell Signaling Technology) as primary antibody and anti-rabbit IgG, HRP-linked antibody (diluted 1:10,000; #7074; Cell Signaling Technology) as secondary antibody. Immunoblots were detected and analyzed with Immobilon Forte (Merck) and the ChemiDoc Imaging System (BioRad).

The whole-cell lysate was generated with radio-immunoprecipitation assay (RIPA) lysis buffer, and the protein concentration was measured by Bio-Rad (Hercules, CA, USA) protein assay following the manufacturer's instructions. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride (PVDF) membranes, and the blots were blocked and incubated overnight at 4°C with the appropriate primary antibody. The following antibodies which obtained from Cell Signaling Technologies (Beverly, MA, USA) were used in this study: anti-SYK antibody (#80460), anti-NF-κB p65 antibody (#8242), anti-IκBα antibody (#4814), anti-ERK1/2 antibody (#4696), anti-p38 antibody (#8690), anti-p-SYK antibody (#2710), anti-p-NF-κB-p65 antibody (#3033), anti-p-IκBα antibody (#2859), anti-p-ERK1/2 antibody (#4370), and anti-p-p38 antibody (#4511). Both the anti-TOSO antibody (#ab56487) and anti-BCL-2 antibody (#ab13-8800) were obtained from Abcam, Cambridge, UK. The blots were then incubated with a specific HRP-conjugated secondary antibody (CST#7074 and 7076, Beverly). Immunodetection was performed by enhanced chemiluminescence.