Figure 8.

TGF-β1/Smad signaling in mesangial cells from Lama1^CKO and control mice. A and B: TGF-β1 mRNA expression and secretion in mesangial cells. MCs were cultured for 48 hours and serum starved for an additional 48 hours before total mRNA extraction and culture media collection. A: TGF-β1 mRNA expression levels measured by RT-qPCR. The bar graph represents the ratio between the TGF-β1 and GAPDH levels. B: Immunoreactive TGF-β1 synthesis measured by ELISA. C: Western blot analysis of TβRI protein levels in MCs. D and E: Smad2 phosphorylation determined by using Western blot analysis. The intensity of signal was analyzed by Multi Gauge version 3.0 (Fujifilm). Relative activity, expressed as the ratio of activated phospho-Smad2/total Smad2/3, was quantified as the fold increase relative to non-stimulation conditions (defined as 1). MCs from control and Lama1^CKO mice were cultured in the absence or presence of 5 μg/mL LM-111 for 24 hours. D: After serum starvation for 24 hours, cells were treated with 10 ng/mL TGF-β1 (WAKO) for 30 minutes. MCs from control and Lama1^CKO mice were serum starved for 24 hours, followed by 1-hour treatment with increasing concentrations of SB-431542 (0.1, 1, 5, and 10 μmol/L) or dimethyl sulfoxide (DMSO), and then stimulated for 30 minutes with 10 ng/mL TGF-β. E: Loss of Smad2 phosphorylation is observed in an SB-431542 dose-dependent manner for both control (dark gray bars) and Lama1^CKO (light gray bars) cells. Data represent means ± SEM. ^∗∗P < 0.01 versus control; ^†P < 0.05 versus laminin-1 treatment cells; ^‡P < 0.01 versus control cells.