Fig. 8. Lactacystin-induced accumulation of CDCrel-1 and ubiquitinated proteins in the centrosome. HEK293 cells transfected with myc-tagged CDCrel-1 were treated without (-Lac; A-C,G-I) or with (+Lac; D-F,J-L) lactacystin (10 µM for 12 hours) and co-stained with antibodies against -tubulin (-tub; A,D) and myc-tag (myc; B,E), or anti--tubulin (-tub; G,J) and anti-ubiquitin (Ub; H,K), respectively. Merged confocal images showed that CDCrel-1 (F and C) and ubiquitinated proteins (L and I) accumulated in the centrosome after lactacystin treatment. Scale bars: 5 µm. Similar results were obtained from at least five sets of confocal images taken from two to three coverslips at each condition. (M) Anti-myc western blot of lysates from cells transfected without (-) or with (+) myc-CDCrel-1 to show expression of the protein and specificity of the antibody (only one band at the correct size was recognized). (N) Anti-ubiquitin western blot of lysates from cells treated with 0 µM, 1 µM, or 10 µM lactacystin (Lac) for 12 hours. Most of the ubiquitin signals represented polyubiquitinated proteins with very little free ubiquitin running at 8.5 kDa (^*).

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