Use of the RUSH system with imMKCLs and PF4 as the α-granule cargo. Differentiated imMKCLs were transfected with a RUSH bicistronic plasmid containing PF4-SBP-Cherry as a cargo-reporter, and their α-granules were labeled with endocytosed and chased fibrinogen–Alexa Fluor 488. Live cell confocal fluorescence microscopy images were acquired before and after the addition of biotin over a 5-hour period. (A) Quantification of the cargo-reporter percent colocalization with fibrinogen–Alexa Fluor 488 is represented with red bars (n = 5). The reciprocal colocalization of fibrinogen–Alexa Fluor 488 with the cargo-reporter is represented with green bars. (B) Images of the cargo-reporter and fibrinogen–Alexa Fluor 488 for one representative cell over a 5-hour period. Note that the cargo-reporter takes several hours to reach α-granules after leaving the ER.