Western Blot Analysis

The cells were lysed using sonication in a radioimmunoprecipitation assay buffer, and the total protein concentration was examined by a protein assay kit (Bio-Rad Laboratory, Hercules, CA, USA). Western blot analysis was performed using the Jess^TM Simple Western system, an automated capillary-based size separation technique (ProteinSimple, San Jose, CA, USA) (39). The proteins of interest were separated and quantified using the 12-230-kDa and 66-440-kDa Jess separation modules (SM-W004 and SM-W008, ProteinSimple) according to the manufacturer's instructions. The primary and secondary antibodies used in these studies are listed in Table 1. The chemiluminescent detection was achieved using peroxide/luminol-S (ProteinSimple). The chemiluminescent images of separated proteins in the capillary were acquired with Compass Simple Western version 5.0.1 software (Build 0911, Protein Simple) that automatically measured the area (chemiluminescence intensity). The results are shown as electropherograms reflecting the peak of the chemiluminescence intensity and as a lane view from the chemiluminescence signal detected in the capillary. The relative density was calculated by dividing the peak area of the protein of interest by the peak area of β-actin.