Fig. (5) Co-localization of PCSK9 with LDLR. A. 14-13-E4 cells were labeled with 0.2 µM Alexa Fluor 488 (Green) as in Fig. (4). B. 14-13-E4 cells were labeled with 5 mM TMR (Red) as in Fig. (3). Following fixation and permeabilization, cells were counter-stained for LDLR and subcellular organelle markers (red in A, green in B). The images were acquired with an Evotech Opera Confocal High Content Imager with appropriate fluorescence filters. Intracellular PCSK9 and LDLR co-localized in the ER and Golgi complex at early time points, and co-localized in lysosomes at later time points. Secreted/internalized PCSK9 mostly co-localized with LDLR in endosomes and lysosomes.