Fig. 6.

NOX activity is necessary for PDGF signaling. a Serum-starved WT- or TKO-MDFs were stimulated with PDGF-BB (50 ng ml⁻¹) for the indicated times. Lysates were subjected to immunoblotting with the indicated antibodies. Representative immunoblots are shown from one of four experiments. See also Supplementary Fig. 5a. Graph at right shows percentage change in phosphorylation of the indicated PDGFRβ tyrosine residues in WT- and TKO-MDFs at the indicated times post-stimulation, quantified from immunoblots (n = 4). Error bars represent SD. b Serum-starved WT- or TKO-MDFs were stimulated with PDGF-BB (50 ng ml⁻¹) for the indicated times. Lysates were subjected to immunoblotting with anti-pMEK1/2 and anti-MEK1 antibodies. Representative immunoblots are shown from one of four experiments. The graph shows the relative phosphorylation of MEK1, quantified from the immunoblots (n = 4), with the value in WT-MDFs at 5 min after PDGF stimulation set to 1. Data represent means ± SD. c Serum-starved WT- or TKO-MDFs were pre-treated with SHP099 (5 μM) or vehicle for 30 min, and stimulated with PDGF-BB (50 ng ml⁻¹) for 2.5 min. Lysates were subjected to immunoblotting with the indicated antibodies. Representative immunoblots are shown from one of four experiments. The graph shows the relative phosphorylation of the indicated PDGFRβ tyrosine residues, quantified from immunoblots (n = 4), with the values from untreated WT-MDFs set to 1. Data represent means ± SD. Values for each phosphotyrosine residue in TKO-MDFs with or without SHP099 treatment were compared by paired two-tailed t-tests. d Schematic of PDGF-evoked redoxosome formation and SHP2 oxidation. See text for details