FIGURE 8.

Specific recruitment of RhoGEFs by PATJ after LMW-HA stimulation of HPMVEC. ECs were serum-starved for 1 h in EBM-2 media and then incubated with LMW-HA (100 nm) or Ephrin-A1-Fc (10 ng/ml) for 5, 15, and 30 min. Cell lysates were prepared in 1% Nonidet P-40 lysis buffer and incubated with anti-PATJ-cross-linked beads for immunoprecipitation. The resulting immune precipitations (IP) were then immunoblotted (IB) with Dbs, LARG, PDZ RhoGEF, Syx, p115 RhoGEF, and PATJ antibodies. Only Dbs was recruited to PATJ in a time-dependent manner with both LWM-HA and Ephrin-A1 treatment of HLMVEC. Panel B, graphical depiction of quantity of protein normalized to PATJ staining intensity (arbitrary units) from experiments described in panel A performed in triplicate and quantitated using computer-assisted densitometry. The asterisks (*) indicates a statistically significant difference (p < 0.05) from control.