Fig. 1

Proteasomal blockade induces Ate1 perinuclear clustering. a Immunofluorescence of CHO-K1 cells transfected with pEGFP (GFP) and pEGFP-N2-Ate1-1 (Ate1-1) and incubated with 10 μM MG132 for 12 h. Immunostaining was performed and analyzed using confocal microscopy. Ate1-1 (green) was detected using an antibody against the GFP epitope, and poly-Ub (pUb, red) proteins were identified using a specific antibody that did not cross-react with mono-ubiquitinated proteins. Nuclei (blue) were stained with Hoechst dye. Pearson's correlation coefficient (r) is provided. (a', a”) (right panel) Insets show higher magnification of the selected regions (white dashed box). (left panel) Histogram of Ate1-1 or GFP and poly-Ub signal intensity levels along the dashed line indicated in the inset. b GFP protein levels in CHO-K1 cells transfected with pEGFP-N2-Ate1-1 (Ate1-1) and treated with 10 μM MG132 (0, 12 h). Ate1-1 protein level was detected using an antibody against the GFP epitope. Actin was used as a loading control. Quantification of Ate1-1 levels at 12 h relative to 0 h is shown. Statistical significance was analysed using two-sided unpaired t-test. Quantifications are representative of two independent experiments and correspond to mean ± SE. ***P≤0.001. c Ate1 KO^Ate1-1 cells were incubated with 10 μM MG132 for 16 h. Subcellular fractionation was performed using a protein separation kit according to the manufacturer's instructions. Whole lysate (WL) and extracts of cytosolic proteins (Cyt), membrane-associated proteins (Memb) from the ER, Golgi, mitochondrial and plasma membrane, soluble nuclear proteins (Nuc), chromatin-bound proteins (Chrom) and cytoskeleton-associated proteins (Cytos) were obtained. Ate1-1 level were detected using an antibody against Ate1. Gapdh and γ-Tubulin (γ-Tub) were used as markers for cytosolic and cytoskeleton-associated proteins, respectively. The total protein marker was used as a loading control. Scale bars: 10 μm (main images), 1 μm (inset)