Ras Activation Assay and Western Blot Analysis

For pull-down of activated GTP-Ras 20 × 10⁶ cells per experiment were starved in IMDM 2% HI-FCS (BMMC) or RPMI 1% HI-FCS (BMMØ) for 3 h. After starvation, cells were resuspended in Hank’s Balanced Salt Solution (15 mM HEPES, 140 mM NaCl, 5 mM KCl, 2.8 mM NaHCO₃, 1.5 mM CaCl₂, 1 mM MgCl₂, 0.06 mM MgSO₄, pH = 7.4) supplemented with 1% BSA and 60 mM Glucose. Stimulation with 4 µM adenosine (Sigma 01890) or 10 nM murine recombinant C5a (R&D Systems 2150-C5-025) was terminated after 2 min by placing tubes on ice, spinning down, and removing supernatant. Subsequently cells were incubated in 1 ml lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM EGTA, 1% NP-40) supplemented with protease inhibitors (Leupeptin, Pepstatin, Aprotinin, PMSF, DTT, DFP) and containing GST-tagged Ras-binding domain of Raf1A (20 µg/reaction) and 100 µM GDP. After 10 min incubation on ice, lysates were centrifuged at 14,000 g for 15 min. For total lysis aliquots (input), 80 µl of the lysate was mixed with 20 µl 5× sample buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 10% β-mercaptoethanol, 20% glycerol and bromophenol blue) before denaturation (96°C, 7 min) and protein separation by SDS-PAGE, and transferred to a PDVF membrane (Immobilon FL, Millipore IPFL0010).

The remaining lysate was incubated with 40 µl 50% Glutathione-Sepharose 4B bead slurry in lysis buffer (GE Healthcare, 17-0756-01) for 2 h at 4°C. Beads were resuspended in 20 µl 2× sample buffer; denatured and pulled-down Ras protein was subjected to SDS-PAGE immunoblotting. The list of western blot antibodies is provided in the supplementary section (Table S3).

Recombinant p84 and p101 kindly provided by R. Williams were used as standards to quantify expression levels of PI3Kγ adaptor proteins on western blots.