![Figure 8. Effect of exogenous RhoA on PLD activity and RhoA translocation in a PMN reconstituted system. (A) PMNs were labeled with [3H]-lyso-PAF. PMNs were incubated with 10 μM C2-ceramide (C2), then disrupted, and membrane was separated from cytosol as in Figure 4. Samples were incubated with 1% ethanol in the presence of 100 μM GTPγS and increasing concentrations of recombinant His-tagged RhoA (rRhoA). [3H]Phosphatidylethanol was separated by using thin-layer chromatography. U indicates unstimulated. (B) Exogenous RhoA translocation to the plasma membrane was determined by Western blot of the membrane pellets with antibody against RhoA in untreated cells (U), control cells with exogenous RhoA (rRhoA), and C2-ceramide (C2)–treated cells with exogenous RhoA. nRhoA indicates native RhoA. Results are representative of 3 experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/103/6/10.1182_blood-2002-11-3341/6/zh80060458160008.jpeg?Expires=1722706655&Signature=GoKat~uElvSLHe66bxCQGtahfUISs7r1mSPeX3jBGaL~j4MSUvRTQcPonu3NVNE3nmJkBz8qslqBnLVsPugjlTxUl~GThJbWX1shY~dAMkNxYJEIX9EOcCYSCK9AigyXPpVx7Ih88H4kN3n3WTYbITJooF~xmFzwI2yLu-X~w9aBqkOAechZQf5jTPVWlSCfQ8sZzCHUPAt2Ja3KoGlSG0iiD4Xb2GOsSauQ00lbA9iMda26eB5yFhOEpR-kh4XlTESpmRXX6uhysgnSp3tj302I1ZwYfVJ7Q6drDYmRQ2JBm1I59jcCHoW1uoBoTME7CVzvMPTI-dKtMkvfzaDhcw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of exogenous RhoA on PLD activity and RhoA translocation in a PMN reconstituted system. (A) PMNs were labeled with [3H]-lyso-PAF. PMNs were incubated with 10 μM C2-ceramide (C2), then disrupted, and membrane was separated from cytosol as in Figure 4. Samples were incubated with 1% ethanol in the presence of 100 μM GTPγS and increasing concentrations of recombinant His-tagged RhoA (rRhoA). [3H]Phosphatidylethanol was separated by using thin-layer chromatography. U indicates unstimulated. (B) Exogenous RhoA translocation to the plasma membrane was determined by Western blot of the membrane pellets with antibody against RhoA in untreated cells (U), control cells with exogenous RhoA (rRhoA), and C2-ceramide (C2)–treated cells with exogenous RhoA. nRhoA indicates native RhoA. Results are representative of 3 experiments.
