Figure 3

Activation of NF-κB by lapatinib involves rapid IκBα turnover. (A-D) Whole cell lysates from parental and lapatinib-selected cells of HER2-positive SkBr3 (A) and BT474 (B) cell lines and triple–negative MDA-MB-231 (C) and MDA-MB-468 (D) cell lines breast cancer cells were harvested and subjected to Western blot analysis with specific antibodies for phosphorylation and protein expression of IKKα/β, IκBα, and tubulin. (E,F) SkBr3 (E), MDA-MB-231 (F), and their lapatinib-treated clones were treated with 25 μM cycloheximide for 0, 0.5 and 1 hours. The protein expression of IκBα was examined by Western blot analysis and quantified. (G,H) SkBr3/Lap (G), MDA-MB-231, and 231/Lap#2 (H) cells were treated with 1 μM MG-132 for 0, 0.5 and 1 hours. The protein expression and phosphorylation of IκBα were examined by Western blot analysis. (I,J) Total RNA was extracted from parental and lapatinib-resistant clones of SkBr3 and MDA-MB-231 cells (I) and from SkBr3/Lap#6 and 231/Lap#2 cells infected with lentivirus of Luc or p65 shRNA for three days (J). The expression of IκBα mRNA was analyzed by RT-qPCR. (K) MDA-MB-231 and 231/Lap cells were infected with lentivirus of Luc or p65 shRNA for three days. The protein expression of IκBα was analyzed by Western blot analysis. shRNA, short hairpin RNA.