Figure 5
From: The autophagy scaffold protein ALFY is critical for the granulocytic differentiation of AML cells
ALFY contributes to retinoid-induced lysosomal protein degradation. (a) Control and ALFY knockdown NB4 cells were treated with 1 µM ATRA or acitretin and long-lived protein degradation assays were carried out at day 4. Where indicated, bafilomycinA1 was added to inhibit lysosome dependent protein degradation. Radioactivity was determined by liquid scintillation counting of at least 3 independent experiments. The percentage of total proteolysis is shown. (b) The panel contains the data of the long-lived protein degradation assays as in A. The results are expressed as bafilomycinA1 (BafA) sensitive proteolysis which was calculated by subtracting the value of the BafA treated sample from the corresponding single agent treatment. (c) HEK293Tcells over-expressing PML-RARα were subjected to immunoprecipitation using anti-ALFY or the corresponding IgG antibody as a control. Western blot (WB) analysis of the immunoprecipitated proteins (IP) and of the total cell lysate (TCL) was performed using RARα (to detect PML-RARα at 110 kDa) and ALFY antibodies. Tubulin was used as a loading control. (d) HEK293T cells were transfected with control RNA, ULK1- or ALFY-targeted siRNA followed by transfection with PML-RARA cDNA. The different cell lines were subjected to starvation for 4 hours before western blot analysis of PML-RARα (~110 kDa) was performed. Tubulin is shown as a loading control. (e) The immunoprecipitation of NB4 cell lysates with anti-ALFY or the corresponding IgG antibody is shown. The Immunoprecipitated proteins (IP) and the total cell lysate (TCL) were subjected to western blot analysis for PML-RARα (110 kDa, using RARα antibody), RARα (~50 kDa) and ALFY using Tubulin as a loading control.
