Rac1 activation assay

Isolation of lipid rafts was performed as described in Rodgers and Rose [46 (link)]. Briefly, 100 × 10⁶ cells were lysed with 1% TritonX-100 Lysis Buffer (10 mM Tris-HC1 pH 7.5, 150 mM NaCI, 5 mM EDTA, 1 mM Na₃VO₄, and proteases inhibitors) and Dounce homogenized. Lysates were centrifuged for 5 min at 1300 g for nuclei and debris removal. For equilibrium centrifugation cleared lysates were diluted in an SW41 centrifuge tube with an equal volume of 85% w/v sucrose in TNEV (10 mM Tris-HC1 pH 7.5, 150 mM NaC1, 5 mM EDTA, and 1 mM Na₃VO₄), overlaid with 6 ml of 30% w/v and 3.5 ml 5% w/v sucrose solutions in TNEV. Samples were centrifuged for 17 h at 200000 g at 4 °C with no brake during the deceleration phase. Eleven density gradient fractions were collected and the flotillin-1 positive fractions were further assessed for Rac1 activity with the Rac1 activation assay (Cell Biolabs, Inc., San Diego, CA) according to the manufacturer’s instructions. To determine expression of the lipid-rafts marker monosialotetrahexosylganglioside (GM1), fractions were dot blotted onto a nitrocellulose membrane. The membrane was saturated with TBS 5% BSA for 2 h and incubated with HRP-conjugated cholera toxin B (1/20,000) (Sigma). Dots were revealed by chemiluminescence.