Involvement of the proteasome in PML/RAR degradation. (A) NB4 cells were treated with 1 μmol/L RA in the presence (+LLnL) or in the absence (n.t.) of 50 μmol/L LLnL. The total lysates were resolved by SDS-PAGE and then analyzed by Western blotting using an anti-RAR antibody. The apparent degradation product of ∼97 kD is shown by the arrowhead. (B) NB4.007/6 cells were treated with 50 μmol/L LLnL (+LLnL) or left untreated (n.t.). Lysates were blotted with anti-RAR antibody. A polypeptide comigrating with PML/RAR appeared after 9 and 12 hours of LLnL incubation (black arrow). (C) NB4 and NB4.007/6 cells infected with retroviruses expressing GFP alone (pinco) or GFP-PML/RAR (pinco-GFP-PR LTR) and were then analyzed by fluorescence microscopy for GFP localization. After 36 hours from infection with the PINCO-GFP-P/R LTR, the NB4 and NB4.007/6 cell lines showed the typical nuclear microspeckled pattern of PML/RAR (panels a and c). Both cell lines showed a diffuse distribution of the GFP protein upon infection with the PINCO control retrovirus (only shown for the NB4.007/6 cells; panel e). Panels b, d, and f show the corresponding DAPI staining. (D) NB4 and NB4.007/6 cells were infected with the pinco or the pinco-GFP-PR LTR retroviral vectors and variably treated with RA or LLNL, as indicated. Corresponding lysates were resolved by SDS-PAGE and then analyzed by Western blotting using anti-RAR, anti-GFP, and anti-β–tubulin (to normalize for protein amounts loaded) antibodies, as indicated.