pAktser473 is elevated and phosphatase and tensin homolog lost in basal-like but not luminal-like xenografts. (A) Immunostaining of luminal-like (LL; top) and basal-like (BL; bottom) xenograft tumor sections from the vehicle control group, stained with anti-pAktser473 (red) and anti-pan-Akt (green) antibodies, and secondary antibodies conjugated with near-infrared (NIR) fluorescent dyes (red channel, pAktser473; green channel, pan-Akt). Negative control sections were stained with secondary antibodies alone (left two images in the panels). Tumors were isolated 3 days after the start of the treatment and 5 hours after the last supplementation of the respective drugs. All scale bars = 1 mm. (B) Quantification data from NIR immunofluorescence imaging (n = 4). The signal intensity of the total Akt immunostaining is similar in BL and LL xenografts, whereas the signal for pAkt ser473 is approximately fivefold higher in BL compared with LL xenografts. **P <0.0005. (C) Immunoblot analysis of the level of pAktser473, total Akt and phosphatase and tensin homolog (PTEN) in lysates from two different BL and LL cancers. Level of β-actin included as a loading control. Numbers above the images and the lanes in the immunoblot refer to the individual tumor-bearing mouse. (D) Higher phosphatidylinositol 3-kinase (PI3K) signaling activity in BL xenografts was also confirmed by immunoblot analysis of pAktthr308 levels.