Detection of LRRK2-mediated Rab phosphorylation in neutrophils isolated up to 24 h after venesection.
Blood was drawn from three healthy donors and left unprocessed at room temperature. At the indicated times, neutrophils were isolated from the blood and treated with or without MLi-2 at 100 nM for 30 min. (A) Neutrophils were then lysed and 10 µg of whole cell extract subjected to quantitative immunoblot analysis with the indicated antibodies (all at 1 µg/ml antibody) and the membranes were developed using the Odyssey CLx scan Western Blot imaging system. Similar results were obtained in two independent experiments. (B) Quantitation of immunoblots was undertaken by analysing phospho-Thr73 Rab10/total Rab10 ratio (top panel) and phospho-Ser935 (full length)/total LRRK2 (full length) ratio (bottom panel).