GDF-15 and TGF-β1 inhibit chemokine-induced activation of Rap-1 via Cdc42. (A) PMNs were either untreated (-) or treated with 100 ng/mL TGF-β1 for 20 minutes and/or treated with 100 ng/mL CXCL1 for 1 minute, followed by isolating activated GTP-bound Rap-1, total Rap-1, and immunoblotting. Minimal (GDP) and maximal (GTPγS) activation levels of Rap-1 are shown on the right. (B) PMNs from either WT or Cdc42^LysMKO mice were untreated (-) or treated with either 100 ng/mL TGF-β1 or 100 ng/mL hGDF-15 for 20 minutes, and/or treated with 100 ng/mL CXCL1 for 1 minute (as indicated), followed by isolating activated GTP-bound Rap-1, total Rap-1, and immunoblotting. Quantification of band intensities (for 3 experiments) is given below. (C) PMNs from either WT or Cdc42^LysMKO mice were treated with TGF-β1, GDF-15, and CXCL1 as above, before binding to soluble ICAM-1-Fc (1.5 μg/1 × 10⁶ cells) and α-human IgG-APC antibody (0.75 μg/1 × 10⁶ cells) was determined by flow cytometry. (D-E) Cdc42 activation after 20 minutes of TGF-β1 or GDF-15 treatment (100 ng/mL) (where indicated) in ALK-5^lox/lox and ALK-5^LysMKO PMNs (D), and TGF-βRII^lox/lox and TGF-βRII^LysMKO PMNs (E) was analyzed with the Cdc42 G-LISA activation assay. Data were read at 490 nm. The depicted experiments in (A-B) represent 1 of 3 independent experiments with similar results. In (C), data are pooled from 5 independent experiments and in (D-E) from 3 independent experiments. Data are shown as mean ± SEM. *P < .05; **P < .01 ***P < .001. GDP, guanosine diphosphate; GTPγS, guanosine 5′-O-[gamma-thio] triphosphate.