Inhibition of lysosomal degradation promotes the localization of MPL and CRT_Del52 in LAMP1 compartments. HEK293T cells transfected to express human MPL along with either CRT_WT or CRT_Del52 were either left untreated or treated with 100 nM BafA1 for 4 hours at 37°C, fixed, and immunostained for LAMP1 (green), MPL (red) and either CRT_WT (anti-CRT[N]) or CRT_Del52 (anti-CRT[C_mut]) (blue) proteins. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). (A) Representative Airyscan microscopy images (n = 2) showing a single z-slice of untreated and treated HEK293T cells expressing specific proteins and stained with specific antibodies, as indicated. Zoomed view of the inset is shown and arrowheads point to the CRT_Del52 and MPL puncta colocalizing within the LAMP1 compartments. Scale bar is 5 μm in all the images except the zoomed inset images in which the scale bar is 2 μm. (B-F) Graphs show quantification of CRT puncta (B), MPL puncta (C), fraction of MPL puncta colocalizing with CRT (D), fraction of CRT puncta colocalizing with LAMP1 structures (E), and fraction of LAMP1 structures colocalizing with CRT or MPL (F) in BafA1-treated HEK293T cells expressing either CRT_Del52 (82 cells from 2 experiments) or CRT_WT (91 cells from 2 experiments). Data were quantified using the segmentation and analysis platform CellProfiler. Graphs were plotted using GraphPad Prism and statistical significance was determined using unpaired t tests.