Fig. 7. Upregulation of TGFß signaling in Man1^GT/GT MEFs. (Aa-i) Immunofluorescence staining of TGFß-treated or untreated wild-type (wt) and Man1^GT/GT MEFs with an antibody to SMAD2/3 (a-c). Cells were infected with a Flag-tagged MAN1 retroviral vector; Flag-positive cells are shown in insets, Flag-negative cells are indicated by arrows (d-f). Dapi was used to show nuclei (g-i). (a) In unstimulated MEFs SMADs2/3 are distributed between the nuclei and cytoplasm. (b) Following treatment with TGFß1 SMADs2/3 accumulate in the nucleus. (c) In Man1^GT/GT MEFs, SMAD2 shows a strong nuclear localization in the absence of TGFß1 treatment. (b,e) In wild-type MEFs, overexpression of MAN1 reduces SMADs2/3 accumulation (inset). (c,f) In the Man1^GT/GT MEFs, MAN1 similarly redistributes SMADs2/3 to the cytoplasm (inset). Scale bar: 20 µm. (B) Western blot of SMAD2 concentrations in the nucleus and cytoplasm. Upper panel: following TGFß1 treatment, wild-type (+/+) and Man1^GT/GT (GT/GT) MEFs were fractionated into nuclear and cytoplasmic extracts and immunoblotted with an antibody to SMAD2. Both TGFß1 untreated and treated Man1^GT/GT cells show enhanced nuclear accumulation of SMAD2. Bottom panel: immunoblot of nuclear and cytoplasmic fractions prepared from Man1^GT/GT and wild-type MEFs expressing Flag-MAN1 showing a marked reduction in the nuclear accumulation of SMAD2. (C) Western blot of MEF whole cell lysates with antibodies to SMAD2 (upper) and SMAD1 (lower). SMAD2, but not SMAD1 is hyperphosphorylated in Man1^GT/GT MEFs following treatment with TGFß1. (D) Man1^GT/GT MEFs show higher levels of both basal and stimulated SMAD transcriptional activity. Man1^GT/GT and wild-type MEFs were transfected with a TGFß-responsive reporter plasmid p3TP-Lux and co-transfected with pTK-RL. Transfected cells were stimulated with indicated concentrations of TGFß1 and analyzed for luciferase activity. Mean±s.e.m.; n=3. (E) Real-time PCR analysis of TGFß target genes. MEFs were treated with TGFß1 and quantitative PCR analysis performed on duplicate extracts. Means±s.e.m. are indicated. (F) Western analysis shows upregulated fibronectin (upper) and p21waf1 (lower) protein levels in TGFß1 treated Man1^GT/GT MEFs. Actin was used as a loading control.