TGF-β protein levels are enhanced in STAT5A/B-null MEFs. (A) Changes in the pSTAT5 and STAT5B levels in control and STAT5-null MEFs before and after GH stimulation as detected by Western blot analysis. (B) Control and STAT5-null MEFs were seeded on a 96-well culture plate, maintained in 15% FCS (left) or 3% FCS (right), and viable cells were assessed by the WST colorimetric assay. Data are mean ± SD. (C) Quantification of total collagen in cell lysates of MEFs (left) or in culture medium (right). Primary MEFs (bars 1 and 4) or MEFs after 10 passages in the absence (bars 2 and 5) or presence (bars 3 and 6) of TGF-β antibody were used. Neutralization antibody was added to the medium every passage (final concentration, 100 ng/ml). Data are mean ± SD. The asterisks indicate significant differences (left, P = 0.024 [left] or P = 0.011 [right]; right, P = 0.021 [left] or P = 0.024 [right]). (D) Changes in TGF-β levels and STAT3 activation in primary MEFs and MEFs after 10 passages before and after GH stimulation as detected by Western blot analysis. (E) Changes in TGF-β levels of cell lysates in control and STAT5-null MEFs before and after TGF-β or IL-6 stimulation as detected by Western blot analysis. (F) Expression levels of TGF-β1, β2, and β3 mRNA in both cells before and after stimulation as detected by real-time RT-PCR analysis. The relative expression level of untreated sample of control MEFs was considered as 1, and the fold expression level of each sample was calculated. Data are mean ± SD. (G and H) Changes in TGF-β levels in control (two lines were used) and STAT5-null MEFs before and after CHx or ammonium chloride treatment. The intensity of protein bands was measured by AlphaImager (Alpha Innotech). The relative intensity level of the untreated sample of each MEF was considered as 1, and the intensity level of each sample was calculated. Three independent experiments were performed and representative data are shown. Recombinant murine TGF-β protein (Wako Chemicals USA, Inc.) was used as a positive control (H, right).