Figure 4.
Inhibition of lysosomal acidification rescues cell surface levels of MPL protein more significantly in the presence of CRTDel52. (A-C) Murine Ba/F3-MPL control (EV) cells or those expressing CRTDel52 were treated with 100 nM BafA1 for 4 hours in media with IL-3. Untreated cells are included for comparison. The surface MPL levels were detected using flow cytometry. (A) Representative histograms of the surface MPL levels with and without BafA1 treatment in both cell lines. (B) Average MFI for surface MPL levels after BafA1 treatment plotted as a ratio to the levels in untreated Ba/F3 cells. One-sample t tests were used for determining the statistical significance. (C) Comparison of BafA1-mediated rescue of surface MPL in control (EV) and CRTDel52 cells. The P value was calculated using a paired t test analysis. Panels B and C include data from 12 experiments from 4 independent transductions of BaF3-MPL cells. (D- E) Ba/F3-MPL control (EV) and Ba/F3-MPL CRTDel52 cells were treated with inhibitors of proteasomal degradation, MG132 (D) or bortezomib (E) at the indicated concentrations for 4 hours at 37°C in media with IL-3. Surface MPL levels measured by flow cytometry are plotted as a ratio of MFI values in treated relative to untreated cells (n = 3). Multiple paired t-tests were used for determining the statistical significance. (F) Representative blots (n = 5) for MPL in lysates of Ba/F3-MPL (EV), Ba/F3-MPL-CRTWT, or Ba/F3-MPL-CRTDel52 cells as indicated, treated with 21 μM MG132, 100 nM BafA1, or both. The immature form of MPL protein (I) and 2 distinct mature forms, M1 (partially mature MPL expressed in CRTDel52 cells) and M2 (detected in CRTWT-expressing cells), are indicated within the MPL blot. Ubiquitin and p62 were probed as markers to show successful inhibition of proteasomal and lysosomal degradation, respectively. (G) Bar graphs show quantification of total, immature, and mature/partially mature MPL proteins in Ba/F3-MPL CRTDel52 cells treated with MG132 and/or BafA1 normalized to the values in untreated cells based on immunoblots. ImageJ was used for the quantification of blots. One-sample t test was used to determine the statistical significance. Graphs were plotted using GraphPad Prism. ns, nonsignificant.

Inhibition of lysosomal acidification rescues cell surface levels of MPL protein more significantly in the presence of CRTDel52. (A-C) Murine Ba/F3-MPL control (EV) cells or those expressing CRTDel52 were treated with 100 nM BafA1 for 4 hours in media with IL-3. Untreated cells are included for comparison. The surface MPL levels were detected using flow cytometry. (A) Representative histograms of the surface MPL levels with and without BafA1 treatment in both cell lines. (B) Average MFI for surface MPL levels after BafA1 treatment plotted as a ratio to the levels in untreated Ba/F3 cells. One-sample t tests were used for determining the statistical significance. (C) Comparison of BafA1-mediated rescue of surface MPL in control (EV) and CRTDel52 cells. The P value was calculated using a paired t test analysis. Panels B and C include data from 12 experiments from 4 independent transductions of BaF3-MPL cells. (D- E) Ba/F3-MPL control (EV) and Ba/F3-MPL CRTDel52 cells were treated with inhibitors of proteasomal degradation, MG132 (D) or bortezomib (E) at the indicated concentrations for 4 hours at 37°C in media with IL-3. Surface MPL levels measured by flow cytometry are plotted as a ratio of MFI values in treated relative to untreated cells (n = 3). Multiple paired t-tests were used for determining the statistical significance. (F) Representative blots (n = 5) for MPL in lysates of Ba/F3-MPL (EV), Ba/F3-MPL-CRTWT, or Ba/F3-MPL-CRTDel52 cells as indicated, treated with 21 μM MG132, 100 nM BafA1, or both. The immature form of MPL protein (I) and 2 distinct mature forms, M1 (partially mature MPL expressed in CRTDel52 cells) and M2 (detected in CRTWT-expressing cells), are indicated within the MPL blot. Ubiquitin and p62 were probed as markers to show successful inhibition of proteasomal and lysosomal degradation, respectively. (G) Bar graphs show quantification of total, immature, and mature/partially mature MPL proteins in Ba/F3-MPL CRTDel52 cells treated with MG132 and/or BafA1 normalized to the values in untreated cells based on immunoblots. ImageJ was used for the quantification of blots. One-sample t test was used to determine the statistical significance. Graphs were plotted using GraphPad Prism. ns, nonsignificant.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal