Herceptin down-regulates HER2 receptors and decreases HER3 phosphorylation, but it does not decrease HER2 phosphorylation.

<p>(A) SKBR3 cells were lysed for Western blot analysis after pre-treatment with 40 μg/ml Herceptin for a period of 4 h, 2 d, or 10 d. Equal amounts of protein were loaded in each lane, and multiple parallel SDS-PAGE gels were run. Membranes were blotted with appropriate antibodies to assess the indicated proteins (shown here and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002414#pbio.1002414.g002" target="_blank">Fig 2E</a>). Note that only one representative actin control is shown for this experiment. (B) SKBR3 cells were grown in 24-well plates and left to grow for at least 24 h before being treated with 40 μg/ml Herceptin for different durations, as illustrated. The viable cells were counted in a cell viability analyzer using trypan blue to stain the dead cells. (C) SKBR3 cells were incubated with either donor alone (HER2-Cy3b) or donor and acceptor (HER2-Cy3b+pHER2-Cy5) to assess HER2 phosphorylation by FRET after pre-treatment with 40 μg/ml Herceptin for different durations, as illustrated. (D) BT474 cells were lysed for Western blot analysis after pre-treatment with 40 μg/ml Herceptin for a period of 1 h, 1 d, or more than 8 months (m) with replacement every week. Equal amounts of protein were loaded in each lane. Membranes were blotted with antibodies for total and phosphorylated HER2 and actin. (E) BT474 cells were lysed for western blot analysis for the indicated proteins after 40 μg/ml Herceptin treatment for 1 hour. Note that other treatment conditions were assessed at the same time in other lanes of the same gel but they were cut from the membranes as they were irrelevant for these blots. The images were spliced from the same gel with a solid line is included to indicate the splice. (F) SKBR3 cells were lysed after 40 μg/ml Herceptin treatment or stimulation with heregulin 100ng/ml for the indicated durations. Equal amounts were loaded on an SDS gel, and membranes were probed for the indicated proteins.