Immunoblotting of Cerebellum Proteins

Whole cerebellum protein samples were prepared using RIPA buffer, sonicated three times on ice, and supernatants were collected after centrifugation. The resulting cerebellum protein extracts were separated on acrylamide gels, transferred to PVDF membranes, and proteins were detected using standard immunoblotting techniques. The following antibodies were used: AMPKα1 (Bethyl Laboratory^®, Montgomery, TX, USA; Cat# A300-507A), AMPKα2 (Bethyl Laboratory^®, Cat# A300-508A), pACC1 (Cell Signaling^®, Danvers, MA, USA; Cat# 11818), β-actin (Cell Signaling^®, Cat# 5125), and goat α-rabbit IgG (Jackson Immunoresearch Laboratory^®, West Grove, PA, USA; Cat# 111-035-045). The CNBP antibody [34 (link)] was a gift from Dr. Gianluca Canettieri, Sapienza University of Rome, Italy. The intensities of Western blot signal bands were quantified using Gel Analysis in ImageJ.