Anti phospho jnk antibody

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).

Proteins were obtained from HK-2 cell lysates and animal tissue, using RIPA buffer (Thermo, USA) containing Complete, Mini Protease Inhibitor Cocktail (Roche, USA) and Halt Phosphatase Inhibitor Cocktail (Thermo, USA). To examine the expression of proteins, separated protein in SDS/PAGE (gradient gels) were transferred onto polyvinylidene difluoride membranes (PVDF) (Bio-rad, USA), and then incubated with 5% BSA (sigma) for blocking. The membranes were incubated with anti-GAPDH antibody (Santacruz, USA), anti-type 1 collagen antibody (abcam, UK), anti-fibronectin antibody (abcam), anti-α-SMA antibody, anti-Vimentin antibody, anti-Lin28a antibody, anti-phospho-ERK antibody, anti-ERK antibody, anti-phospho-JNK antibody, anti-JNK antibody, anti-phospho-p38 antibody, anti-p38 antibody, anti-phospho-smad3 antibody and anti-smad3 antibody (Cell signaling, USA). After incubating with HRP-linked antibody (Cell signaling), the protein expression on blots was detected by ChemiDoc^TMXRS+ (Bio-rad) and the bands were quantified using the Image Lab^TM Software (Bio-rad).