Figure 6.

Attachment induces the phosphorylation of cPLA₂ on the membrane independent of Ca²⁺-mediated translocation. HeLa cells in suspension were treated without (A and B) or with (C and D) 1 μM ionomycin or 20 μM BAPTA-AM (E and F) for 10 min. At this concentration of ionomycin the Ca²⁺ influx across the plasma membrane was detected and measured in the HeLa cell system (Chun and Jacobson, 1992). Likewise, the concentration of BAPTA-AM used in these experiments has been shown to chelate [Ca²⁺]_i in HeLa cells (Chun and Jacobson, 1992). Cells incubated without ionomycin (A and B) were homogenized in buffer either lacking (A) or containing (B) calcium. Cells treated with ionomycin (C and D) or BAPTA-AM (E and F) were either left in suspension (C and E) or allowed to attach and spread on gelatin for 15 min (D and F). Samples C–F were extracted in Ca²⁺-free buffer to prevent artificial translocation. After separation of the cell lysates into a soluble cytosolic fraction (C) and NP-40-soluble membrane fraction (M), 60 μg of protein were electrophoresed per lane, and the electroblots were probed with mouse anti-human cPLA₂ IgG. A representative gel of three experiments is shown.