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. 2013 Sep 11;3:2623. doi: 10.1038/srep02623

Figure 1. Ub levels are downregulated by the knockdown of Ubb mRNA with siRNA.

Figure 1

(a) Schematic representation of the 4 Ub genes; Rps27a and Uba52 encode Ub as a fusion protein with the ribosomal subunits S27a and L40, respectively, whereas Ubb and Ubc encode tandem units of Ub with 3 and 9 repeats, respectively. All Ub gene products are known to be processed co-translationally. (b) SH-SY5Y neuroblastoma cells were transfected with 20 nM control siRNA or Ubb siRNA for 48 h, and the mRNA level of each Ub gene was analyzed by PCR. Ubb siRNA effectively and specifically depleted Ubb mRNA. β-actin mRNA was used as a control. (c) SH-SY5Y cells were transfected with different concentrations of Ubb siRNA (5, 10, and 20 nM) for 48 h, and the Ub level was analyzed by Western blotting following SDS-PAGE of whole protein extracts. A mono-Ub (mUb) can be resolved from conjugated Ubs appeared as smear and several discrete bands including ubiquitinated histone (arrow). (d) The amounts of mono-Ub and conjugated Ub were independently compared by densitometry (n = 2). Both decreased in a dose-dependent manner, but the downregulation of mono-Ub was particularly noticeable. The data are shown as the mean ± SD, #, p < 0.1; *, p < 0.05; **, p < 0.01.