Parkin acts upstream of Rag GTPases to regulate TFEB subcellular localization. (A and B) HeLa cells stably expressing TFEB-GFP with (B) or without (A) mCherry-Parkin were transfected with empty vector or active or inactive RagB/D heterodimer DNA (detected with αHA antibody). The indicated cells were starved (2 h) or treated with DMSO or O/A (6 h) before fixation and immunofluorescence analysis. Images are representative of n = 2 experiments. 75–100% of cells observed (mean of 175 cells per condition) exhibited the given phenotype. Asterisks indicate αHA-negative cells. Bars, 10 µm.