Figure 4

Translocation of CAP1 upon agonist stimulation. (A) Reduced membrane-associated CAP1 upon thrombin stimulation. Platelets (8 × 10⁸/mL) were treated with 0.1 U/mL thrombin in the presence of 1 mM EGTA to prevent aggregation for the indicated times prior to lysis and fractionation. Fractions were normalised by volume and resolved on 12% SDS-PAGE, blotted onto PVDF membrane and probed with antibodies for the indicated proteins. CD36, membrane marker; Syk, cytosolic marker. Membrane-associated CAP1 was quantified by densitometry, normalised to CD36 and expressed relative to CAP1 in the 0 s membrane fraction. Data represent mean ± SEM of four independent experiments. (B) CAP1 moves away from the high speed (HS) detergent insoluble pellet of thrombin stimulated platelets. Platelets (8 × 10⁸/mL) were stimulated with 0.1 U/mL thrombin prior to lysis in Triton X-100 containing buffer and spun at high speed (100,000 × g) for 1 hour. Only pellets of HS samples were resolved on 12% SDS-PAGE gel, blotted onto PVDF membrane and probed with antibodies for CAP1 and β-actin. CAP1 was quantified by densitometry, normalised to β-actin and expressed relative to the 0 s pellet fraction. The low speed (LS) (15,600 × g) pellet shows the typical increase in actin upon thrombin stimulation. Data represent mean ± SEM of four independent experiments. (C) Effect of indomethacin and apyrase on the response of CAP1 in the HS detergent insoluble pellet upon thrombin stimulation. The experiment was performed as in (B) in the presence or absence of indomethacin (10 µM) and apyrase (1 U/mL) using the indicated thrombin doses for 1 min. Blots of HS pellets were probed with antibodies for CAP1 and β-actin. The experiment was repeated with centrifugation at low speed (LS) to show the effect of thrombin on actin. CAP1 was quantified by densitometry, normalised to β-actin and expressed relative to the basal pellet fraction. Data represent mean ± SEM of three independent experiments. (D) Effect of collagen and resistin on the amount of membrane-associated CAP1. The experiment was performed as in (A) using 10 µg/mL collagen for 3 min or 200 ng/mL resistin for 15 min. Only the membrane fractions were analysed. Integrin β3 was used as membrane marker and for normalisation. Data represent mean ± SEM of 4 to 6 independent experiments. For all panels *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 relative to 0 s or basal. Mann-Whitney test was used for non-parametric data and t-tests were used for parametric data with a Bonferroni correction where appropriate. Full-length blots are presented in Supplementary Fig. 3.