Western Blot Analyses

CD11c magnetic beads-purified C57BL/6 mice derived BMDCs (2×10⁶ cells) were treated with 1 µg/ml LPS or 4 mg/ml Huaier extractum for 0, 15, 30, and 60 minutes, respectively. After treatment, DCs from different groups were harvested and lysed with lysis buffer. The lysates were centrifuged at 12,000× g for 20 minutes at 4°C, the supernatants were harvested and the protein concentrations were determined by BCA assay. Protein samples were fractionated on 12% Tris-glycine gels, followed by proteins transfer onto a PVDF membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat dry milk and then probed with 1:500 to 1:1,000 diluted primary antibodies against the following mouse proteins (Cell Signaling Technology, Beverly, MA, USA), including Akt (Cat#4685), phosoho-Akt (Cat#4060), PI3K (Cat#4249), JNK (Cat#9252), phospho-JNK (Cat#4668), p38 (Cat#9212), phospho-p38 (Cat#4511), ERK (Cat#4695), phospho-ERK (Cat#4370), and GAPDH (Cat#51332), followed by HRP-labeled secondary antibodies at a 1:5,000 dilution. Antibody binding was visualized with a chemiluminescent substrate and visualized on autoradiography film.