Colocalization images for mutated di-lysine variants in combination with mutated di-arginine motif variants in HEK293T cells. This figure shows representative colocalization images of single immunostained HEK293T cells expressing the Lys159Ala + Lys161Ala, Lys159Ala + Lys161Ala + Arg98Ala + Arg100Ala, Lys159Ala + Lys161Ala + 98_100delRTR, and the Lys159Ala + Lys161Ala + p.Arg98Trp mutant hVKORC1 variants, respectively. Each panel is composed of 4 images; the first one is a merged picture of the green-colored hVKORC1 variant (EGFP tagged), the red-stained ER (primary antibody: anti-PDI; secondary antibody: AlexaFluor 594), and the nucleus counterstained with ToPro3 (blue). Colocalization is represented with increasing intensity of yellow. The second image shows cells expressing the EGFP-tagged VKORC1 protein only, followed by the third image showing the red-stained ER. The last column shows colocalized regions as white dots using the colocalization highlighter plug-in embedded in ImageJ 4.3. All images were rendered using ImageJ version 4.3. The di-lysine–mutated hVKORC1 variant Lys159Ala + Lys161Ala shows a higher amount of ER retention than the di-arginine variants shown in Figure 2. Also, in combination with the di-arginine variants the di-lysine variants do not show a more drastic decrease in ER retention.