Immunoblotting Procedure Modifications

Immunoblotting procedures were modified from Lee et al. [13 (link), 15 (link)] with a few modifications. Aliquots containing 20 μg of sample homogenates were heated with denaturing buffer at 60°C for 15 min. The samples were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels. The separated proteins were transferred to 0.45 μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) using a tank transfer system (Mini protean 3; Bio-Rad, Hercules, CA, USA). The membranes were pre-incubated for 2 h in PBS containing 0.2% (v/v) Tween 20 and 5% (w/v) nonfat dried milk to minimize nonspecific binding. Next, the blots were incubated at 4°C overnight with antibodies (Table 2). The membranes were then incubated at room temperature for 1 h with secondary antibodies (Table 2). Blots were developed using Immobilon^TW Western (Millipore). Signals were obtained using the Chemidoc XRS+ image system (Bio-Rad), and then the data were analyzed with Image Lab software (version 3.0; Bio-Rad). The results were converted to numerical values to compare the relative protein abundance of the immunoreactive bands.