Figure EV3. DRiPs that accumulate upon proteasome inhibition or temperature upshift are ubiquitinated and do not colocalize with nuclear speckles (related to Fig 3).

1. Subcellular distribution of DRiPs and SC35, used as a marker for nuclear speckles, in HeLa cells that were left untreated or treated as indicated. Scale bars: 10 μm.
2. Immunoprecipitation (IP) of puromycylated proteins from nucleolar or nucleoplasmic extracts. HeLa cells were treated as indicated for 2 h (25 μM puromycin, 20 μM MG132) before fractionation. Western blots against puromycin and ubiquitin are shown.
3. HeLa cells were treated with puromycin (5 μg/ml) for 1 h, followed by recovery for 6 h in drug‐free medium (−) or in the presence of MG132 (10 μM) or NH₄Cl (20 mM). Cytoplasmic and nuclear proteins were fractionated. Clearance of puromycylated and ubiquitinated proteins were analyzed by immunoblotting in both fractions. TUBA4A and LMNB1 were used as cytoplasmic and nuclear loading controls.