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. 2005 Apr 11;169(1):179–189. doi: 10.1083/jcb.200501098

Figure 7.

Figure 7.

Tyrosine phosphorylation of c-Src and caveolin-1 in sulfatide-loaded fibroblasts. (a) MEFs were loaded with gal-sulfatide and treated with 10 μg/ml Lm-1. Equal protein loads of cell lysates were analyzed in immunoblots. Transient Src activation (PY416) was detected within 30 min after Lm-1 treatment. (bottom left) Ratio of Src-PY416/total Src. (b) Lm-1 does not induce Src phosphorylation in fibroblasts in the absence of sulfatide loading. Fibroblasts with or without sulfatide loading were incubated with 10 μg/ml Lm-1 for 1 h. Cell lysates were immunoblotted with either c-Src-Py416 or c-Src–specific antibodies. (c) αDG antibody and Lm-1 fragment E3 inhibit Src phosphorylation in sulfatide-loaded fibroblasts treated with Lm-1. Gal-sulfatide–loaded fibroblasts were treated with 10 μg/ml Lm-1 for 1 h in the presence of either 100 μg/ml BSA,100 μg/ml E3, 250 μg/ml E8, 10 μg/ml mouse IgM, 10 μg/ml IIH6, or 10 μg/ml of β1-integrin antibody Ha2/5; lysed; and immunoblotted for pSrc and c-Src. (d) DG expression is required for Lm induction of Src activation in sulfatide-loaded fibroblasts. Fibroblasts derived from wild-type or DG-null embryonic stem cells treated with gal-sulfatide were incubated with 10 μg/ml Lm-1 for 1 h and analyzed for pSrc and total Src. (e) Ablation of the β1-integrin gene does not prevent Lm-1–induced Src phosphorylation in sulfatide-loaded fibroblasts. β1-integrin–deficient fibroblasts (GD25) and β1-integrin–transduced GD25 control cells were treated the same as described in d, with lysates analyzed for pSrc and total Src. (f and g) Lm-1 assembly on sulfatide-loaded fibroblast surfaces does not require DG or β1-integrin. DG-null (f) and β1-integrin–null (g) fibroblasts, loaded with gal-sulfatide and incubated with 10 μg/ml Lm-1 for 1 h, were fixed and immunostained for Lm α1. (h) Caveolin-1 phosphorylation is induced by Lm-1 in sulfatide-treated embryonic lung fibroblasts. Fibroblasts were treated the same as described in panel a, and analyzed for Py14-caveolin-1 (Cav-1). The densitometry plot (caveolin-1-Py14/total caveolin-1) is also shown. (i) Src inhibition decreases Lm-induced caveolin-1 phosphorylation. Sulfatide-loaded fibroblasts were treated with Lm-1 plus Src kinase inhibitor PP2 (2 μM) or SU6656 (2 μM) for 1 h. Cell lysates were analyzed in immunoblots for caveolin-1-Py14 (Cav-1-Py14) or total caveolin-1 (Cav-1).