Figure 7. Spatiotemporal activation of Rap1 reduces association of FLN with β₂.

(a) RalGDS-RBD mCherry in control BAF cells stimulated with CXCL12 as indicated. Scale bar, 5 μm. (b) FRET-based Rap1 activity sensor-expressing control BAF cells stimulated with CXCL12 as indicated. A ratio image of mTurquoise/CFP represents FRET efficiency. Scale bar, 5 μm. (c) Control cells expressing RalGDS-RBD mCherry perfused on the endothelium with CXCL12. The digital images of contact areas of the cells taken under shear flow. Scale bar, 5 μm. (d) COS cells transfected with T7-Rap1V12, Rap1N17 or αL and β₂ were lysed and pulled down using GST fusions of FLNa repeats 1–3, 4–6, 7–10 or 21, and immunoblotted with anti-T7 or anti-β₂. (e; Upper) Lysates from COS cells transfected with T7-Rap1V12 or Rap1N17 immunoprecipitated with anti-FLNa, and immunoblotted with anti-T7. (Lower) Lysates from Cos cells transfected with Halo-FLNa wild-type and the mutants deleting repeat 1(Δ1), 2 (Δ2) or 3(Δ3), and T7-Rap1V12, precipitated using HaloLink Resin, and immunoblotted with anti-T7 or Halo. (f) Lysates from COS cells transfected with Halo-FLNa, T7-Rap1V12 or N17, and αL and mRFP-β precipitated with HaloLink resin, and immunoblotted with anti-mRFP, T7, Halo and αL. (g) Membrane fraction from BAF/LFA-1 cells transfected with vector or T7-Rap1V12 pulled down using the cytoplasmic region of β₂-GST fusion protein. Bound FLNa and total FLNa and T7-Rap1V12 were analysed using anti-FLNa or anti-T7 antibody. (h) Lysates from BAF/LFA-1 cells transfected with vector or Spa-1, ±CXCL12 for 1 min, immunoprecipitated with anti-β₂, and immunoblotted with anti-FLNa. (i; Left upper) Western blots of total cell lysates from BAF/LFA-1/L-selectin cells transduced with scramble (Control) or FLNa or b-targeting shRNA. Actin is a loading control. (Left lower) Epitope expression of monoclonal antibody KIM127. Data are normalized against LFA-1 expression detected by TS1/18. *P<0.01 versus control. (Centre) Control or FLNa/b KD cells perfused on LS12 monolayers (n=150). *¹P<0.02, *²P<0.01 versus frequency of control cells. (Right) Effects of anti-L-selectin or anti-LFA-1 antibody on the adhesion events (n=150 per each group). *P<0.01 versus frequencies of untreated FLNa/b KD cells. Data represent the means±s.e.m. of triplicate experiments.