Rabbit anti gapdh pab

The western blotting procedure was previously described [13 (link)]. Membranes were incubated with rabbit anti-CAP1 mAb (EPR8339(B); Abcam) or rabbit anti-GAPDH pAb (Santa Cruz Biotechnology), with a dilution of 1:1000 (CAP1) and 1:100 (GAPDH), the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit antibody) was added (at a dilution of 1:2000), and an incubation was performed for 3 h using the iBind Flex Western System (Thermo Fisher Scientific). Protein bands were detected with the enhanced Novex® ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) using LAS-3000 (Fujifilm).

Kidneys and livers were homogenized in homogenization buffer (10 v/w) containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, and 25 mM sodium fluoride, supplemented with protease inhibitor mixtures (Thermo Scientific) and phosphatase inhibitor mixtures (Thermo Scientific). 20 μg proteins were loaded onto 10% SDS-polyacrylamide gels. Following electrophoresis, proteins were transferred to an Immobilon -P membrane (Millipore, USA). Membranes were blocked for 1 hour with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST). Membranes were probed with the primary antibodies diluted in 5% BSA in TBST. The dilution for each antibody was as follows: rabbit anti-LRRK2 mAb (Epitomics), 1 : 3000; rabbit anti-GAPDH pAb (Santa Cruz), 1 : 2000; and rabbit anti-HIF-2α mAb (Santa Cruz), 1 : 2000. After washing with TBST, the blots were incubated with secondary antibodies (Jackson ImmunoResearch) conjugated to horseradish peroxidase. Immunoreactive bands were detected by the chemiluminescence reagent (ECL) (GE Healthcare, UK).