Figure 5.
Inhibition of lysosomal degradation rescues CRTDel52 levels, whereas proteasomal pathway inhibition promotes CRTDel52 degradation. (A-D) Representative immunoblots (n = 2) showing CRTDel52 levels (A,C) in the lysates of control (EV) or CRTDel52 expressing Ba/F3-MPL cells treated for 4 hours with different concentrations of MG132 (A) or bortezomib (C). Bar graphs (B,D) show quantification of CRTDel52 band intensities from blots of Ba/F3-MPL CRTDel52 cells (n = 2) treated with different concentrations of MG132 (B) or bortezomib (D), normalized to the CRTDel52 band intensities in untreated cells. (E) Representative histograms of surface CRT detected by flow cytometry using anti-CRT(Cmut) antibody in Ba/F3-MPL, Ba/F3-CRTDel52, and Ba/F3-MPL CRTDel52 cells. (F) The bar graph shows the MFI of surface CRT, detected by anti-CRT(Cmut) antibody, on Ba/F3-MPL CRTDel52 cells treated with 21 μM MG132 and/or 100 nM BafA1, normalized to the MFI values for untreated cells (n = 5). (G-I) Ba/F3-MPL, Ba/F3-CRTDel52, or Ba/F3-MPL CRTDel52 cells as indicated were treated with 21 μM MG132, 100 nM BafA1, or both drugs for 4 hours at 37°C in media with IL-3. Representative blots (n = 5) showing the levels of cellular CRTDel52 (G). Graphs show band intensities of CRTDel52 quantified from immunoblots of lysates from drug–treated Ba/F3-CRTDel52 or Ba/F3-MPL CRTDel52 cells normalized to the band intensities of untreated cells (H) or paired comparisons of band intensities from MG132-treated cells relative to those of cells treated with MG132 + BafA1 (I). (J) Representative blot of secreted CRTDel52 IP with anti-CRT(Cmut) antibody from the cell culture media of Ba/F3-CRTDel52 cells (n = 2) and Ba/F3-MPL CRTDel52 cells (n = 6) that were either untreated or treated with 21 μM MG132 and/or 100 nM BafA1 for 4 hours at 37°C in media with IL-3. (K-L) Quantification of secreted CRTDel52 band intensities from IP/immunoblots of drug-treated cells, normalized to the values for untreated cells (K). Line graphs show secreted CRTDel52 band intensities from MG132-treated cells compared to those of cells treated with MG132 + BafA1 (L). Each line in graphs I and L represents an individual experiment and lines connect data points for the indicated treatments within the experiments. CRTDel52 (A,C,G,J) was probed using an anti-CRT(Cmut) antibody. GAPDH blots in panels A,C,G show equal loading of lysates in the different lanes. ImageJ was used for the quantification of blots. Graphs were plotted using GraphPad Prism. Statistical significance was determined using one-sample t tests in panels B,D,F,H,K and paired t tests in panels I,L. P value <0.05 are shown. The bands marked by asterisk in panels A, C and G are non-specific bands. ns, nonsignificant.

Inhibition of lysosomal degradation rescues CRTDel52 levels, whereas proteasomal pathway inhibition promotes CRTDel52 degradation. (A-D) Representative immunoblots (n = 2) showing CRTDel52 levels (A,C) in the lysates of control (EV) or CRTDel52 expressing Ba/F3-MPL cells treated for 4 hours with different concentrations of MG132 (A) or bortezomib (C). Bar graphs (B,D) show quantification of CRTDel52 band intensities from blots of Ba/F3-MPL CRTDel52 cells (n = 2) treated with different concentrations of MG132 (B) or bortezomib (D), normalized to the CRTDel52 band intensities in untreated cells. (E) Representative histograms of surface CRT detected by flow cytometry using anti-CRT(Cmut) antibody in Ba/F3-MPL, Ba/F3-CRTDel52, and Ba/F3-MPL CRTDel52 cells. (F) The bar graph shows the MFI of surface CRT, detected by anti-CRT(Cmut) antibody, on Ba/F3-MPL CRTDel52 cells treated with 21 μM MG132 and/or 100 nM BafA1, normalized to the MFI values for untreated cells (n = 5). (G-I) Ba/F3-MPL, Ba/F3-CRTDel52, or Ba/F3-MPL CRTDel52 cells as indicated were treated with 21 μM MG132, 100 nM BafA1, or both drugs for 4 hours at 37°C in media with IL-3. Representative blots (n = 5) showing the levels of cellular CRTDel52 (G). Graphs show band intensities of CRTDel52 quantified from immunoblots of lysates from drug–treated Ba/F3-CRTDel52 or Ba/F3-MPL CRTDel52 cells normalized to the band intensities of untreated cells (H) or paired comparisons of band intensities from MG132-treated cells relative to those of cells treated with MG132 + BafA1 (I). (J) Representative blot of secreted CRTDel52 IP with anti-CRT(Cmut) antibody from the cell culture media of Ba/F3-CRTDel52 cells (n = 2) and Ba/F3-MPL CRTDel52 cells (n = 6) that were either untreated or treated with 21 μM MG132 and/or 100 nM BafA1 for 4 hours at 37°C in media with IL-3. (K-L) Quantification of secreted CRTDel52 band intensities from IP/immunoblots of drug-treated cells, normalized to the values for untreated cells (K). Line graphs show secreted CRTDel52 band intensities from MG132-treated cells compared to those of cells treated with MG132 + BafA1 (L). Each line in graphs I and L represents an individual experiment and lines connect data points for the indicated treatments within the experiments. CRTDel52 (A,C,G,J) was probed using an anti-CRT(Cmut) antibody. GAPDH blots in panels A,C,G show equal loading of lysates in the different lanes. ImageJ was used for the quantification of blots. Graphs were plotted using GraphPad Prism. Statistical significance was determined using one-sample t tests in panels B,D,F,H,K and paired t tests in panels I,L. P value <0.05 are shown. The bands marked by asterisk in panels A, C and G are non-specific bands. ns, nonsignificant.

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