P-selectin is an SNX17 cargo and traffics through the plasma membrane in a COMMD3-dependent manner. (A) Spinning disk confocal fluorescence microscopy images of imMKCL cells expressing P-selectin WT or F826G-GFP and Cherry-SNX17 or SNX17-Cherry (DN). Bar = 5 µm. (B) Colocalization analysis of the cells shown in A. Statistical significance was determined via unpaired, 2-tailed Student t test (P-selectin WT+Cherry-SNX17, n = 25; P-selectin WT+SNX17-Cherry [DN], n = 29; P-selectin F826G+SNX17-Cherry [DN], n = 11). DN, dominant negative. (C) Confocal fluorescence microscopy images of WT and COMMD3 KO imMKCL cells that were incubated with primary antibodies against P-selectin, CD71, and SNX17 and subsequently fixed, permeabilized, and stained with species-specific secondary antibodies conjugated with Alexa Fluor 488, 546, and 647, respectively. To help visualization, the edge of some cells has been drawn with a dotted line. Bar = 5 µm. (D) Quantification of fluorescence intensity of internalized anti-P-selectin, CD71, and SNX17 antibodies in WT and COMMD3 KO imMKCL cells. Statistical significance was determined via unpaired, Mann-Whitney U test (WT, n = 99; COMMD3 KO, n = 76). While detection of SNX17 is negligible in both samples, it was slightly higher in COMMD3 KO cells. A.U., arbitrary units.