Figure 2

UBA52 regulates protein synthesis.

(A) Ubiquitin expression in UBA52-deficient cells. Total RNA was isolated from DLD-1 cells 24 h after short interfering RNA (siRNA) transfection. UBC, UBB, UBA52, and UBA80 mRNA levels were measured by quantitative real-time reverse-transcription PCR and normalized to ACTB levels. Error bars indicate standard deviations. Data represent three independent experiments (n = 8). **P < 0.01 (one-way ANOVA followed by Tukey’s test). (B) UBA52 cleavage in DLD-1 cells. FLAG-UBA52-green fluorescent protein (GFP) expression in DLD-1 cells. DLD-1 cells were transfected with FLAG-GFP or FLAG-UBA52-GFP. After 45 h, cells were lysed and immunoblotted for the indicated proteins. Data are representative of more than three independent experiments. (C) Cleave-resistant mutants in HEK293T cells. HEK293T cells were transfected with three types of mutation vectors. After 24 h, cells were lysed and immunoblotted for the indicated proteins. G75/76A mutations lead to cleavage resistance (FLAG-UBA52-GFP (CR))¹⁷. Data are representative of more than three independent experiments. (D) UBA52 localization in DLD-1 cells. DLD-1 cells were transfected with FLAG-GFP or FLAG-UBA52-GFP. After 24 h, images were acquired using a confocal laser microscope. Data are representative of three independent experiments. GFP (green) and 4′,6-diamidino-2-phenylindole (blue). (E) Subcellular fractions of DLD-1 cells. DLD-1 cells were transfected with a UBA52 siRNA and lysed for ultracentrifugation at the indicated time. S100 (cytosol) and P100 (crude ribosome pellet) fractions were immunoblotted for the indicated proteins. Data are representative of more than three independent experiments. (F,G) Protein synthesis in UBA52-deficient cells. DLD-1 cells (F) or Hela cells (G) were transfected with UBA52 or ribosomal protein (RP) S3 siRNAs. The cells were treated with cycloheximide (100 μg/ml) for 3 h. Cells were incubated with O-propargyl-puromycin (20 μM) for 30 min and then harvested for the protein synthesis assay. Data are representative of more than two independent experiments. *P < 0.05, **P < 0.01 (one-way ANOVA followed by Tukey’s test). We confirmed knockdown efficiency by quantitative real-time (RT) PCR and normalized to ACTB levels. Data are representative of two independent experiments.