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Fig. 4. Expression profile of TWEAK in the mammary gland. (A)
Expression of Fn14 in mammary epithelial cells. Fn14 was expressed in
undifferentiated and 10-day differentiated KIM2 mammary epithelial cells, as
determined by qRT-PCR analysis. (B) Immunoblotting for TWEAK
demonstrated expression of full length (35 kDa) TWEAK in nuclear and
cytoplasmic compartments in KIM2 cells, while soluble (18 kDa) TWEAK was
exclusively cytoplasmic. Nuclear control was retinoblastoma protein (Rb).
(C) DAB immunohistochemistry for TWEAK demonstrated nuclear staining at
all mammary gland developmental time points examined. Cytoplasmic TWEAK
staining was seen late in lactation (10 dl) and early in involution (12 and 24
hours' involution). (D) Confocal analysis of TWEAK localisation in KIM2
cells. Undifferentiated KIM2 cells were fixed and stained for TWEAK, using
AlexaFlour 555. A single slice of the confocal analysis is shown, with nuclei
visualised by DAPI staining. The bottom panel shows a merged image, with
sectioning through a nuclear puncta. (E) Immunoblotting for TWEAK
during mammary gland development showed that the soluble form of TWEAK was
detected predominantly at 24-72 hours' involution. Scale bars: 100 µm in C;
20 µm in D.
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