Anti p p38

Total proteins were extracted from heart tissues or RAW264.7 cell lysates and supplemented with a cocktail of protease inhibitors and phosphatase inhibitors. Proteins were quantified to an equivalent amount, separated by SDS-PAGE gel, and electro-transferred to a PVDF membrane. Having been blocked in 5% skimmed milk, membranes were incubated with antibodies: anti-LFA-1 (ITGAL, Affinity Biosciences. DF5625, 1:1000), anti-p-JNK (Affinity Biosciences. AF3318, 1:1000), anti-total JNK (Affinity Biosciences. AF6138, 1:1000), anti-p-ERK (Affinity Biosciences. AF1015, 1:1000), anti-total ERK (Affinity Biosciences. AF0155, 1:1000), anti-p-p38(Affinity Biosciences. AF4001, 1:1000), anti-total p38 (Affinity Biosciences. AF6456, 1:1000) and GAPDH. The loading control was GAPDH (Proteintech, 60004-1-lg, 1:10000). Western blot bands were analyzed with the ChemiDicTM XRS + Imaging System (Bio-Rad Laboratories, Hercules, CA, USA), and the densities were quantified with Multi Gauge Software of Science Lab 2006 (FUJIFILM Corporation, Tokyo, Japan).

The procession of protein extraction in this study can be referred to the article (Luo et al., 2021 (link)). 20 µg per sample of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and deposited onto a polyvinylidene fluoride (PVDF) membrane after the concentration of proteins was determined. The membranes were blocked with 5% milk for 2 h before incubation with the primary antibody at 4°C overnight, which is as follow, Anti-p-38 (Affinity Biosciences, OH, United States), Anti-p-p-38 (Affinity Biosciences, OH, United States), β-Tubulin (Affinity Biosciences, OH, United States). After response with the primary antibody, the blotted PVDF membrane was treated with horseradish peroxidase (HRP) conjugated secondary antibod (Boster Biological Technology Co. Ltd.). The ECL chemiluminescence western blot detection technique was performed with a gel imaging system and the band intensity were analyzed via ImageJ.