Evaluating MPP13 Expression in Rat Ankles

MPP13 expression was evaluated in rat ankles using Western blot, as previously described [29 (link)]. In RIPA buffer, the frozen ankle joint samples were homogenized with a phosphatase/proteinase inhibitor cocktail. Homogenate was then centrifuged, and the protein concentration was determined using the Bradford reagent. SDS-PAGE 10% gels were used to separate equal amounts of proteins, which were then transferred to PVDF membranes. After blocking with 5% BSA, the membranes were incubated overnight at 4 °C with primary antibodies against MPP13 and β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The membranes were washed and then incubated for 1 h at RT with the appropriate secondary antibody. A CCD camera-based imaging device was used to capture chemiluminescent signals. On a ChemiDoc MP imager, image analysis software was used to read the band intensities of the target proteins against the β-actin by protein normalization.