Figure 1.

Effect of the Downregulation of Ubr-Ubiquitin Ligases of the Arg/N-Degron Pathway In Vitro

(A) Western blot analysis of UBR1, UBR2, UBR4, and UBR5 proteins in Hepa 1–6 and AML-12 cells after 72 h of exposure to 1 nM siRNA. (B) Quantification of protein Ubr expression in Hepa 1–6 and AML-12 cells after 72 h of exposure to 1 nM siRNA. (C) Asp¹¹¹⁹-BRCA1^f (produced from ^fDHFR-Ub^R48-Asp¹¹¹⁹-BRCA1^f) and Val¹¹¹⁹-BRCA1^f were expressed in Hepa 1–6 cells transfected with siRNA. Protein extracts were analyzed by SDS-PAGE and western blot against FLAG. (D) Analysis of cell proliferation by the neutral red assay on Hepa 1–6 cells after 72 h of exposure to siRNA. (E) Analysis of cell death TUNEL assay in Hepa 1–6 cells after 72 h of exposure to 10 nM siRNA. Red nuclei represent TUNEL-positive cells. Scale bar, 100 μm. (F) Migration assay performed on Hepa 1–6 cells after 72 h of exposure to siRNA against Ubrs. Results show mean ± SD. p values were determined by a Student’s test (*p = 0.01).