Western blotting
Cell lysis samples were run through gel electrophoresis using 10%
SDS-PAGE gels and proteins were electrotransfered onto PVDF membranes
(97062-900, VWR). The membranes were then blocked with 5% dried skimmed
milk in TBS-T (50 mM Tris pH 8.4, 0.9% NaCl, 0.05% Tween-20) for 1
hour and incubated overnight at 4°C in the presence of previously listed
primary antibodies in 5% milk. Prior to incubating the membrane in the
secondary antibody for 1 hour at 25°C, it was washed with TBS-T 3 times
for 10 min. each. The membrane was developed using Radiance Plus
Reagents (AC2103) and chemiluminescent bands were revealed (Azure
Biosystems).