Reduction in total and surface human MPL levels when coexpressed with CRTDel52 in Ba/F3 cells. IL-3-dependent murine Ba/F3 cells were transduced for coexpression of human MPL and either CRTWT or CRTDel52 protein. Ba/F3 cells expressing only human MPL protein (EV) were included as controls. (A) Representative histograms showing surface and total MPL staining detected by flow cytometry in Ba/F3-MPL cells expressing either MPL alone or MPL along with CRTWT or CRTDel52, as indicated. (B) Bar graphs show quantification of MFI values of surface MPL (B) and total MPL (C) measured by flow cytometry in CRTWT or CRTDel52 expressing cells normalized to the MFI values of EV cells within the same experiments. The data points represent measurements taken in 38 (B) and 25 (C) independent experiments using cells from ∼8 different retroviral infections. P values show statistical significance determined by paired t test analyses using GraphPad Prism. (D-E) Representative immunoblots (D, n = 14; E, n = 3) showing the levels of total MPL protein in Ba/F3 cells coexpressing either CRTDel52 or CRTWT compared with EV control cells (D) and without or with Endo H digestion (E). GAPDH is shown as a loading control. The top and bottom bands in the MPL blots represent the mature (M) and immature (I) forms of MPL proteins, respectively. (F-G) Bar graphs show the fraction of immature (F) and mature (G) human MPL protein in EV, CRTWT, and CRTDel52-expressing cells, quantified from the intensities of the mature and immature MPL bands from the immunoblots, normalized to the intensities of GAPDH bands. Image J was used to quantify band intensities. Paired t tests were used in GraphPad Prism to determine statistical significance, as indicated by the P value.
