DCs treated with PLP-2 at 100 μg/mL or LPS at 1 μg/mL for 24 h were collected by centrifugation and washed three times with pre-cold phosphate buffer (PBS). Then total proteins of DCs were prepared using KGP950 Phosphorylated Protein Extraction kit (KeyGEN Biotech, Jiangsu Province, China). Besides, the nuclear and cytoplasmic proteins were prepared using Nuclear and Cytoplasmic Protein Extraction kit (Beyotime, China). Protein concentration was determined using BCA assay kit (Beyotime, China).
Total proteins were separated by electrophoresis using a 10% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. Membrane was blocked using Tris-buffered-saline with Tween-20 (TBST) containing 5% BSA (Solarbio) at 25 °C for 1 h, and incubated with primary rabbit anti-phospho-p38, anti-phospho-ERK or anti-phospho-JNK antibody (All from Cell Signaling Technology, USA) for 12 h at 4 °C. The membrane was then incubated with secondary horse radish peroxidase (HRP)-conjugated anti-rabbit antibody (ZSGB-BIO, Beijing, China) for 1 h at 37 °C. Finally, membrane was developed with enhanced ECL kit (Beyotime, China). Chemiluminescence detection was performed on ChemiDoc XRS+ imaging system (BIO-RAD). Similarly, IκB-α in the cytoplasma extracts were also determined by Western blot analysis with anti-IκB-α antibody (Cell Signaling Technology, USA).
Total proteins were separated by electrophoresis using a 10% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane. Membrane was blocked using Tris-buffered-saline with Tween-20 (TBST) containing 5% BSA (Solarbio) at 25 °C for 1 h, and incubated with primary rabbit anti-phospho-p38, anti-phospho-ERK or anti-phospho-JNK antibody (All from Cell Signaling Technology, USA) for 12 h at 4 °C. The membrane was then incubated with secondary horse radish peroxidase (HRP)-conjugated anti-rabbit antibody (ZSGB-BIO, Beijing, China) for 1 h at 37 °C. Finally, membrane was developed with enhanced ECL kit (Beyotime, China). Chemiluminescence detection was performed on ChemiDoc XRS+ imaging system (BIO-RAD). Similarly, IκB-α in the cytoplasma extracts were also determined by Western blot analysis with anti-IκB-α antibody (Cell Signaling Technology, USA).