Anti phospho nf κb p65

After growth and challenge utilizing a 6-well format (CELLTER, Santiago, Chile), lysis of the cells was accomplished with the RIPA buffer (Solarbio, Beijing, China), followed by electrophoretic shift of the protein samples onto the PVDF (polyvinylidene difluoride) membranes (Solarbio, Beijing, China). The next step was a 1-h blockage of unbound PVDF sites with buffer and subsequent incubation based on optimal primary antibody dilutions, such as anti-phospho-NF-κB p65 & anti-NF-κB p65 (both Bioss, Beijing, China), anti-phospho-IκB alpha (Bioss, Beijing, China), anti-phospho-p38 MAPK & anti-phospho-ERK1/2 MAPK (both Cell Signaling Technology, MA, USA), anti-beta-Actin (Bioss, Beijing, China), anti-phospho-NF-κB p65 (Bioss, Beijing, China), anti-phospho-JNK1 (Bioss, Beijing, China), and anti-GAPDH (Proteintech, Wuhan, China) antibodies, for the proteins of interests for 12 h at 4°C. After rinsing four times with PBS-containing Tween 20 (PBST), a further 1-h incubation of the membranes proceeded with alkaline phosphatase (AP)-labeled secondary antibodies at 37°C. Then, the membranes were rinsed four times in PBST (Solarbio, BeiJing, China). A BCIP/NBT Substrate color kit was used for visualization.