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.