Figure 9.

Tiam1 controls the expression of small GTPase and cytoskeletal regulators, and the association of Rasa3 with Rac in neutrophils adhering to ICAM1. Wild type and Tiam1^–/– neutrophils were allowed to adhere to 50 mm glass coverslips coated with ICAM1 during priming with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 50 min at 37°C, 5% CO₂. DFP was added for 10 min before neutrophils were stimulated with 1.5 µM fMLP for 1 min and then lysed. The cleared supernatants (total lysates) were incubated with immobilised Rac1^G15A to isolate proteins that interact with nucleotide-free Rac. (A) Targeted mass spectrometry was performed for 21 proteins found to bind Rac1^G15A (left). 14 proteins could be compared between wild type (Wt) and Tiam1^–/– samples, 12 proteins in at least 2 out of 3 independent experiments (right). Wt/Tiam1^–/– ratios are mean ± SEM or range, as appropriate, of 2-3 independent experiments; coloured dots depict the different experiments. Statistics are one-way ANOVA with Dunnett’s multiple comparisons test on log-transformed ratios. (B) Total lysates from the experiments in (A) were western blotted with the indicated antibodies. Representative western blots and coomassie loading control are shown. Blots of selected proteins were quantified by densitometry (right). Wt/Tiam1^–/– ratios are expressed as mean ± SEM of 3 independent experiments; statistics are two-way ANOVA with Sidak’s multiple comparisons test on raw band intensities. Bottom panel: The Wt/Tiam1^–/– Rac1^G15A binding ratio is expressed as a function of the Wt/Tiam1^–/– expression level for the indicated proteins.