GRP78 protein ATPase activity assay
RA FLSs were seeded in 10 cm cell culture dishes and transfected with GRP78 recombinant plasmid. After 48 hours of transfection, cells were scraped to extract protein. ATPase activity is determined using Kinase-Lumi™ Chemiluminescence Kinase Activity Detection Kit (Beyotime Biotechnology). Specifically, ATP with different concentrations was diluted by kinase reaction buffer and 50 μl Kinase-Lumi™ Chemiluminescence Kinase Detection Reagent was then added. Following reaction at room temperature (about 25 °C) for 10 minutes, chemiluminescence detection was performed with a multifunctional microplate reader to draw an ATP standard curve. After measuring the value of GRP78 protein substrate, the enzyme activity was calculated according to the standard curve.