Fig. 5

CK2 is important for preserving the stability of SCD-1 in ccRCC cells. (A) Cells were incubated with vehicle (DMSO) or 2.5 µM CX-4945 for 16 h prior to the addition of 100 µg/ml cycloheximide (CHX) for the indicated times. Whole cell lysates were subjected to Western blot analysis. β-actin detection was used as loading control. One representative experiment is shown. Densitometric analysis of SCD-1 protein band signals were plotted against time assigning for each cell line 100% to values corresponding to control cells at time 0 (bar graphs), or to control cells and CX-4945-treated cells, respectively, at time 0 (line graphs). Average values are expressed in percentage +/- STDEV. Experiments were performed at least three times yielding similar results. (B) Cells were incubated with vehicle or 2.5 µM CX-4945 for 16 h prior to the addition of 50 µM MG132 for 6 h. Whole cell lysates were processed as in (A). Detection of phosphorylated heat shock factor 1 (P-HSF-1) at S326 was carried out to verify induction of cellular stress in the presence of proteasome inhibitors [85]. Values from the densitometric analysis of SCD-1 protein band signals are expressed in percentage assigning 100% to values corresponding to cells treated with MG132. Experiments were performed two times. *P < 0.05, **P < 0.005, ***P < 0005