Colocalization images for wt hVKORC1 and mutated di-arginine motif variants in HEK293T cells. This figure shows representative colocalization images of single immunostained HEK293T cells expressing wt hVKORC1, Arg33Ala + Arg35Ala, Arg98Ala + Arg100Ala, the 98_100delRTR variant, and the human p.Arg98Trp mutant variant, respectively. Each panel is composed of 4 images; the first one is a merged picture of the green-colored EGPF-tagged hVKORC1 protein, the red-stained ER (primary antibody: anti-PDI; secondary antibody: AlexaFluor 594), and the blue-counterstained nucleus by ToPro3. Colocalization is represented with increasing intensity of yellow in the first image. The second image shows cells expressing the EGFP-tagged hVKORC1 protein in green only, followed by the third image in which the ER is red stained. 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 images clearly show a high ER colocalization for wt hVKORC1 and the Arg33Ala + Arg35Ala variant. All other variants affecting the Arg98_Arg100 di-arginine motif show a drastic decrease in ER colocalization.