Figure 3.

SPTBN1 induces ubiquitination and degradation of p65. (A and B) Binding of p65 with IκBa kept identical in HCC cells upon the inhibition of SPTBN1. SNU-449 cells were transiently transfected with siRNAs as indicated for 48 h (A) and PLC/PRF/5 cells stably transduced with SPTBN1 shRNA or non-specific (ns) shRNA were cultured for 48h (B). Cells were then subjected to immunoprecipitation (IP) and immunoblotting with antibodies as indicated. Whole cell lysates of above cells (inputs) were analyzed by Western blotting by antibodies as indicated. Data are representative of two to three independent experiments with similar results. The intensities of SPTBN1, p65, IκBα, pIκBα and actin were measured by ImageJ software, and the relative expression level of each protein/actin was generated. (C) PLC/PRF/5 cells were transiently transfected as indicated for 48h. Cells were then treated with cycloheximide (CHX) for the indicated time periods and were analyzed for the stability of p65 by Western blotting (upper). The intensities of p65 and actin were measured by ImageJ software, and the relative expression units of p65/actin were generated and normalized relative to the expression unit in 0 h. The percentage remaining of p65 expression (compare to zero hour) was then calculated (lower). (D) SNU-449 cells were transfected as indicated for 48 h. Cells were then treated without or with 10 µM MG132 for 4 h before cell lysis and analyzed by Western blotting. (E) SNU-449 cells and PLC/PRF/5 cells were transiently transfected as indicated for 48 h. Cells were then treated with 10µM MG132 for 4 h before cell lysis. Cells were immunoprecipitated with IgG or antibody to p65 and the immunoprecipitates were then immunoblotted with anti-ubiquitin antibody to detect ubiquitination of endogenous p65.