Investigating IGF1R and CXCR4 Signaling

The immunoprecipitation experimental procedures were as previously described54 (link). Cultures were treated with recombinant human IGF1 (Invitrogen) at 300 ng/mL80 (link) and/or recombinant human SDF-1α (R&D System) at 100 nM81 (link). First, hDSCs were collected and lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% NP-40, 150 mM NaCl, 0.5% sodium deoxycholate, and protease inhibitor). The cell lysate (300 μg) was incubated with protein A/G-agarose beads at 4 °C for 6 hours. Then, antibodies against IGF1R (Santa Cruz Biotechnology), CXCR4 (R&D System), phosphotyrosine (pY) (p-Tyr-100, Cell Signaling), Giα₂ (T-19, Santa Cruz Biotechnology) and Gβ (M-14, Santa Cruz Biotechnology), or control antibody IgG (anti-hemagglutinin clone F-7, Santa Cruz Biotechnology) were added and reacted for 6 hours at 4 °C. These immunocomplexes were incubated on protein A/G-agarose beads at 4 °C overnight. After three washes with lysis buffer, the immunocomplexes were then examined by western blot with anti-IGF1R, anti-phosphotyrosine, anti-CXCR4 antibodies, anti- Giα₂ and Gβ, or anti-control IgG.