Full-length PI4KIIIα visits the plasma membrane dynamically. (A) Confocal imaging of HeLa cells transfected with GFP-M1*-PI4KIIIα (left) or GFP-M1-PI4KIIIα (right). (B) Anti-PI4KIIIα Western blot comparing electrophoretic mobility of endogenous (Endog.) PI4KIIIα and either exogenously expressed M1-PI4KIIIα or M1*-PI4KIIIα in lysates of HEK cells. Molecular masses are given in kilodaltons. (C) Alignment of the N-terminal region of vertebrate PI4KIIIα orthologues. The black shading denotes conservation of amino acid identity, and the gray shading denotes conservation of amino acid similarity. (D) Ribosome footprinting reveals M1 and T*, but not M1*, as likely PI4KIIIα translation start sites. The data shown were pooled from two biological replicate experiments. (E) TIRF microscopic analysis shows highly dynamic spots of GFP-M1-PI4KIIIα, but not of GFP-M1*-PI4KIIIα, at the plasma membrane, revealing very transient visits of the enzyme to this membrane. COS-7 cells were transfected with GFP-M1-PI4KIIIα (top row) or GFP-M1*-PI4KIIIα (bottom row) and imaged by TIRF microscopy, with a 1-s interval between each image acquisition. Bars: (A) 20 µm; (E) 2 µm.