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. 2011 Jan 31;6(1):e16571. doi: 10.1371/journal.pone.0016571

Figure 6. S1P induces Rac1 activation and IQGAP1 tyrosine phosphorylation in HPAECs.

Figure 6

HPAECs (∼90% confluence) grown on 35-mm dishes were starved for 3 h in 0.1% FBS in EBM-2 without growth factors and treated with S1P (1 µM) for 5 min. Activated Rac1 was immunoprecipitated from total cell lysates (500 µg of total protein) from control and S1P (1 µM) treated cells using PAK-1 PBD agarose beads as described under “Experimental Procedures”. A, Rac-1-GTP bound to PAK-1 PBD were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-Rac1 antibody. B, IQGAP1 was immunoprecipitated from total cell lysates (500 µg of total protein) from control and S1P (1 µM) treated cells using anti-IQGAP1 antibody. Immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-p-Tyr antibody. Shown is a representative blot from three independent experiments.