Figure 4.

IKKα and IKKβ increase Myc protein stability. The stability of Myc protein was analyzed by Western blot analysis of the whole cell lysates prepared from (A) MCF7 with or without a 12 hours, 10 μM Bay11-0782 treatment (B) wild-type and kinase-dead IKKα transfected cells (C) wild-type and kinase-dead IKKβ transfected cells along a time course after adding cycloheximide. The stain of tubulin was used as loading control. The reading of Myc density was normalized to the reading of tubulin density. Each data represents mean ± SD calculated from two independent experiments. Whole cell lysates were prepared from (D) MCF7 cells, (E) wild-type and kinase-dead IKKα transfected cells, and (F) wild-type and kinase-dead IKKβ transfected cells were subjected to coimmuoprecipitation using indicated antibodies. The precipitated complex was further Western blot analyzed the corresponding proteins shown in the figure. (G) Confocal microscopy observation. Breast cancer tissues which were previously identified positive or negative for IKKα, IKKβ and Myc expressions by IHC staining were used. The slides were dual stained with rabbit anti-IKKα/mouse anti-Myc antibodies coupled with FITC-conjugated goat anti-rabbit IgG/Rodamine-conjugated donkey anti-mouse IgG antibodies or goat anti-IKKβ/mouse anti-Myc antibodies coupled with FITC-conjugated donkey anti-goat IgG/Rodamine-conjugated donkey anti-mouse IgG antibodies, respectively.