FIGURE 2.

Structure-function analysis of TSP1-induced EGFR activation. A, schematic of baculovirus-derived recombinant TSP1 domains. B, A431 cells were exposed for 0.5 h to equimolar concentrations (214 nm) of baculovirus-derived recombinant TSP1 domains, including the following: lane 1, NH₂-terminal heparin-binding domain + oligomerization + procollagen domain (NoC); lane 2, procollagen domain + properidin repeats 1-3 (CP123); lane 3, properidin repeat 3 + EGF-like repeats 1-3 (P3E123); lane 4, EGF-like repeats 1-3 (E123); lane 5, EGF-like repeats 1-3 + Ca²⁺-binding repeats + COOH terminus (E123-CaG); lane 6, EGF-like repeat 3 to COOH terminus (E3CaG); and lane 7, COOH terminus (CaG). C, TSP1 (214 nm) was preincubated with either of two antibodies, C6.7 and HB8432, each targeting its EGF-like repeats, or a species- and isotype-matched antibody control, B7-1, after which the TSP1 was incubated with A431 cells. D, A431 cells were exposed for 1 h to equimolar concentrations (214 nm) of E123, E12, E2. or E3 or media alone. A431 cells were exposed for 0.5 h to increasing concentrations of recombinant TSP1 EGF-like repeats (E123) or media alone (E) or exposed for increasing exposure times to a fixed concentration of recombinant TSP1 EGF-like repeats (3.8 μg/ml or 214 nm), a concentration equimolar to 30 μg/ml TSP1, or media alone (F). G, A431 cells transfected with EGFR targeting (lane 2) or control (lane 1) siRNAs were cultured for 48 h, after which they were exposed for 1 h to E123 (214 nm) or media alone. Cells were lysed and processed for immunoblotting with anti-phospho-EGFR (Tyr-1068) antibodies. The blots were stripped and reprobed with anti-β-tubulin antibody to indicate protein loading and transfer. IB, immunoblot; IB^*, immunoblot after stripping; each of these blots are representative of ≥2 independent experiments. H, for each phospho-EGFR Tyr-1068 immunoblot (see Fig. 1A and Fig. 2E), densitometric quantification of each Tyr(P)-1068 signal was normalized to the β-tubulin signal for the same lane on the same stripped and reprobed blot. Vertical bars represent mean (±S.E.)-fold increase of arbitrary densitometry units of Tyr(P)-1068 signal normalized to arbitrary densitometry units of β-tubulin signal, each relative to the simultaneous control. n = 3. ^*, significantly increased compared with the equimolar concentration of E123 at p < 0.05.