![Fig. 6. Failure of C2-ceramide to inhibit p47phox phosphorylation or translocation. / (A) PMNs were labeled with H3[32P]O4, washed, and exposed to 10 μM C2-ceramide or buffer for 30 minutes at 22°C. Cells were then stimulated at 37°C with 50 ng/mL G-CSF (10 minutes) and then 1 μM FMLP (5 minutes) or 100 ng/mL PMA (5 minutes) as indicated. PMNs were lysed with 1% Igepal CA-630, and p47phox was immunoprecipitated. Samples (equal volumes) were run on 10% SDS-PAGE and exposed to x-ray film. One representative experiment of 3. (B) Samples (equal volumes) from panel A were loaded on a 25-cm-tall 10% SDS-PAGE gel to separate p47phox from the 55-kd IgG band. Proteins were transferred to PVDF, and the membrane was probed with anti-p47phox to show equivalent loading. (C) PMNs were treated with C2-ceramide and stimulated as indicated for panel A. Cells were probe sonicated and fractionated on 15% to 35% discontinuous sucrose gradients as described in “Materials and methods” to isolate plasma membrane. Equal amounts of protein were run on 10% SDS-PAGE, and Western blots were probed with antibody against p47phox. One representative experiment of 3. U indicates unstimulated; G, G-CSF; F, FMLP; C2, C2-ceramide; and P, PMA.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/99/4/10.1182_blood.v99.4.1434/6/h80422119006.jpeg?Expires=1735570281&Signature=Gg5mpHiQdbWDT3qTqORyypdXobQVZ1wGG5zOPVgzpFCPOPIvEjJGZsBcya5vaClyF6TPSdaDTYQK5QRTjj~tKppGszwMf7MRi2HeH14-ZxUg~GwJnrhoAjiafdE5HCk9vI1xvb-HTaok-qgrBhkUphrrzL0yNOfmEsf8Cy7ihgTSsljfO~RYZolju4A2iqPxiXWaaG8IJsiSi~vSCCrBHvWo3RrbMGknrQ99Z0KPzb77sMlwfdqh-GAbYGspaAzAhoi4WPAfcDqiRs0pcaRqvDvkvMtwAQwP2jF-F8mAF3Ra5OpO2NeMyyZhLkn8K6D9sk1pGX9QVrCbxJ3zaxCVuw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Failure of C2-ceramide to inhibit p47phox phosphorylation or translocation.
(A) PMNs were labeled with H3[32P]O4, washed, and exposed to 10 μM C2-ceramide or buffer for 30 minutes at 22°C. Cells were then stimulated at 37°C with 50 ng/mL G-CSF (10 minutes) and then 1 μM FMLP (5 minutes) or 100 ng/mL PMA (5 minutes) as indicated. PMNs were lysed with 1% Igepal CA-630, and p47phox was immunoprecipitated. Samples (equal volumes) were run on 10% SDS-PAGE and exposed to x-ray film. One representative experiment of 3. (B) Samples (equal volumes) from panel A were loaded on a 25-cm-tall 10% SDS-PAGE gel to separate p47phox from the 55-kd IgG band. Proteins were transferred to PVDF, and the membrane was probed with anti-p47phox to show equivalent loading. (C) PMNs were treated with C2-ceramide and stimulated as indicated for panel A. Cells were probe sonicated and fractionated on 15% to 35% discontinuous sucrose gradients as described in “Materials and methods” to isolate plasma membrane. Equal amounts of protein were run on 10% SDS-PAGE, and Western blots were probed with antibody against p47phox. One representative experiment of 3. U indicates unstimulated; G, G-CSF; F, FMLP; C2, C2-ceramide; and P, PMA.
