Fig. 1. Analysis of IKK2 expression in mammary glands of floxed mice. (A) Southern blot analysis of Ikk2 locus. Lanes 1-4 controls: lane 1 wild-type DNA; lane 2 DNA from floxed/wild-type tissue (not recombined); lane 3 DNA from floxed/floxed tissue (not recombined); lane 4 DNA from floxed/deleted tissue. Lane 5 empty. Lanes 6-11: mammary gland DNA samples from Cre⁺/Ikk2^fl/fl mice at 24 hours' involution, showing that varying levels of recombination have occurred (lanes 6-11, respectively: 37, 79, 43, 36, 31 and 37% recombination). (B) Protein levels of IKK2 were determined in mammary glands from Cre^-, Cre⁺/Ikk2^fl/wt or Cre⁺/Ikk2^fl/fl mice by immunoblotting. At 24 hours' involution, reduced IKK2 levels were seen in Cre⁺/Ikk2^fl/fl and Cre⁺/Ikk2^fl/wt mice (loading control was -tubulin). (C) EMSA of mammary gland tissue nuclear extracts for NF-B and OCT1. NF-B DNA-binding activity was determined by densitometry and normalised to OCT1. In the absence of IKK2 (Cre⁺/Ikk2^fl/fl), NF-B DNA-binding activity is reduced by approximately 50%. (D) DAB immunohistochemistry for NF-B p50 and p65 subunits in mammary gland sections from Cre^- or Cre⁺/Ikk2^fl/fl mice at 24 hours' involution. Compared to Cre^- glands, nuclear p50 staining was reduced in Cre⁺/Ikk2^fl/fl glands (<). Nuclear p65 staining was weak in Cre^- glands, and undetectable in glands from Cre⁺/Ikk2^fl/fl mice. Percentage of positively staining nuclei is indicated in the lower right corner of each image. Scale bar: 100 µm.