Fig. 3. Changes in gene and protein expression following loss of IKK2. (A) Western blot analysis for total and serine phosphorylated levels of AKT, FOXO3a and of STAT3 at 24 hours' involution in mammary glands from Cre^-, Cre⁺/Ikk2^fl/wt or Cre⁺/Ikk2^fl/fl mice. Significantly increased levels of pAKT and pFOXO3a were seen in the absence of IKK2. Loading control was -tubulin. (B) DAB immunohistochemistry for FOXO3a in mammary gland sections from Cre^-, Cre⁺/Ikk2^fl/wt or Cre⁺/Ikk2^fl/fl mice at 24 hours' involution. Reduced nuclear FOXO3a staining was seen in Cre⁺/Ikk2^fl/fl glands (<). Percentage of positively staining nuclei is indicated in the lower right corner of each image. (C) A representation of previously obtained microarray data showing expression of DR ligands throughout lactation and early involution (Clarkson et al., 2004). (D) qRT-PCR for the DR ligands: FASL, TNF, TRAIL and TWEAK and DR TNFR1. Significant reductions in TWEAK (^** t<0.01), TNF (^* t<0.05) and TNFR1 as determined by the paired t-test, were seen in glands from Cre⁺/Ikk2^fl/fl mice compared with Cre^- control mice. Levels are arbitrary units and have been normalised to cyclophilin A. (E) qRT-PCR for TWEAK of control and 15dPGJ₂ treated undifferentiated KIM2 cells. Statistically significant decreases in TWEAK mRNA as determined by the paired t-test, were seen following 4 hours (^**t<0.01) and 8 hours (^***t<0.005) of 15dPGJ₂ treatment. Levels are arbitrary units and have been normalised to cyclophilin A. Scale bar: 100 µm.