Fig. 3.

Proteasome inhibition leads to the accumulation of polyubiquitinated proteins in the cytosol in a CRM1-dependent manner. (A) Ubiquitinated proteins accumulate in the cytosol on proteasome inhibition and are relocated to the nucleus in response to inhibition of nuclear export. NIH 3T3 cells were transfected with 0.5 μg of HA-ubiquitin and treated with DMSO, 0.5 mM EPX for 8 h, 0.5 mM EPX and 10 nM LMB for 8 h, or CRM1 knockdown for 72 h and 0.5 mM EPX for the last 8 h. The cells were immunostained with anti-HA (12CA5) antibody (green). The white circles indicated by dashed lines indicate the nuclear region. (Scale bar: 10 μm.) (B) Detection of the nuclear export of endogenous ubiquitinated proteins using antiubiquitin antibody. NIH 3T3 cells were treated with DMSO, 0.5 mM EPX for 8 h, 0.5 mM EPX and 10 nM LMB for 8 h, or CRM1 knockdown for 72 h and 0.5 mM EPX for the last 8 h. The cells were immunostained with antiubiquitin (Apu2) antibody (red). The white circles indicated by dashed lines indicate the nuclear region. (Scale bar: 10 μm.) (C) Nuclear export of ubiquitinated proteins in HUVECs. HUVECs were treated with DMSO (Left) or 0.5 mM EPX for 8 h (Right). The cells were immunostained with antiubiquitin (FK2) antibody (green) and antiubiquitin (Apu2) antibody (red). The white circles indicated by dashed lines indicate the nuclear region. (Scale bar: 10 μm.) (D) Quantification of the Apu2 immunostaining of HUVECs (with SD). The total fluorescence intensity of the cytoplasmic region or nuclear region of HUVECs immunostained with Apu2 antibody was quantified (n = 10 experiments; each experiment contains 500 cells). ***P < 0.001.