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. 2023 Jul 31;12(15):1973. doi: 10.3390/cells12151973

Figure 5.

Figure 5

Blockage of DCUN1D1 decreases global neddylation, ubiquitination and expression of neddylation components and shows preferential neddylation activity of cullin proteins in PCa. Western blot analysis of DU145 and DU145 DCUN1D1-KD cells. Protein extracts were obtained from the cells and subjected to Western blot analysis using (a) anti-ubiquitin, (b) anti-Nedd8. Blockage of DCUN1D1 decreased the expression of the neddylation pathway components including (c) the E1 NAE heterodimer APPB1 (top panel) and the neddylation-conjugating enzyme, UBC12 (bottom panel) and (d) the cullin-associated proteins RBX1 (top panel) and CAND1 (bottom panel). Inhibition of DCUN1D1 showed preferential NEDD8 modification of the cullin family of proteins. Immunoblot analysis of DU145 and DU145 DCUN1D1 knockdown cell protein extracts using (e) anti-cullin 1, (f) anti-cullin 3, (g) anti-cullin 4A, (h) anti-cullin 4B and (i) anti-cullin 5. The GAPDH loading control was probed using the anti-GAPDH antibody. Experiments were independently repeated three times and a representative image of an independent experiment is represented.