Fig. 2.
TGF-β1 reduces uPAR cleavage through induced PAI-1 expression. a, f-h. Western blot analysis of whole cell lysates of cultured and stimulated AT84-uPAR cells. Where indicated, cell lysates were either treated with PNGase F (+) or samples received the same treatment except that PNGase F was omitted (−). Glycosylated uPAR is indicated as uPARglc. d-e. Western blot analysis of conditioned medium from equally seeded amounts of AT84-EV and AT84-uPAR cells. a. Cells cultured in FBSM with or without 2 ng/ml active human TGF-β1 for 24 h. b-c. Total RNA from treated (TGF-β1) or untreated (Ctrl) cells was isolated and the relative expression of uPAR mRNA (B) or uPA mRNA (C) was analysed using RT-qPCR. The error bars show the +SD. N = 3. d. Cells cultured in SFM or FBSM and stimulated with 2 ng/ml TGF-β1 for 24 h as indicated and PAI-1 protein levels were analysed. Controls received either no additives (−) or the TGF-β1 buffer (0). Positive control: recombinant mouse PAI-1 (rmPAI-1). e. AT84-uPAR cells cultured in either SFM or in FBSM. Cells were either unstimulated (−) or stimulated (+) with 2 ng/ml TGF-β1 and/or 10 μM of the TGF-β1-inhibitor SB431542 as indicated. f. AT84-uPAR cells were cultured for 24 h in FBSM and stimulated with 2 ng/ml TGF-β1 as indicated. g. Deglycosylated whole cell lysates from AT84-uPAR cells cultured in SFM stimulated with 2 ng/ml TGF-β1 +/− the inhibitor SB431542 as indicated were analysed for uPAR protein levels. h. Cells were cultured for 24- or 48 h in SFM, FBSM or TMEM. Cells were treated with 2 ng/ml TGF-β1 in 10% FBS as indicated. The LRP1 protein is indicated. Arrowhead shows an unknown band