Characterization of leucine-rich repeat kinase-2 (LRRK2) aggregates in transfected 293t cells. Double immunofluorescence was performed with the monoclonal anti-V5 antibody (red) and 1181#1 (green) in 293t cells transfected with LRRK2-V5. 32 hours after transfection, cells were treated with vehicle (A) or MG132 (10 µM) (B) for 16 hours before fixation. (C) Quantitative assessment of cells containing large aggregates, as determined by independent cell counts, comparing control to MG132 treatment. Data represent average ± SD (*, p = 0.004; n = 3). (D) Transfected 293t cells begin forming aggregates within 24 hours after transfection, and (E) perinuclear aggregate formation is blocked by treatment with nocodazole from 12 hours after transfection until fixation at 24 hours post-transfection. Images are representative of 3 independent experiments. Bar scale: 50 µm.