Rab27a controls PM levels of PI(4)P and the CD63-mediated recruitment of PI4KIIα to the PM. (A) LSCM images of live control and Rab27a-silenced Jurkat cells transiently transfected with GFP-P4M-SidM plasmid and observed 24 h later. (B) Representative single-cell intensity profile plots of the mean fluorescence intensity of GFP-P4M-SidM quantified in the selected regions indicated with a white rectangle shown in A. (C) Quantification of the localization of the GFP-P4M-SidM signal predominantly at the PM or the cytosol in control and Rab27a-silenced cells was performed by blinded observers on a per-cell basis, in 200 cells of each condition. (D) LSCM of live control and Rab27a-silenced Jurkat cells transiently transfected with PI4KIIα-GFP plasmid and labeled with LysoTracker red. (E) LSCM of live control and Rab27a-silenced Jurkat cells transiently transfected with PI4KIIα-GFP and mCherry-CD63 plasmids. Manders correlation coefficient map is shown. Pseudocolored scale represents the contribution of each pixel to Manders colocalization coefficient (overlap coefficients for control and Rab27a-silenced cells: 0.74 ± 0.01 and 0.75 ± 0.02, respectively). Transmitted light images are shown on the right images of D and E. KD, knockdown. Bars, 2 µm.