Fig. 9. Phosphorylation-dependent gel shift of cPLA₂ (A) and localization of phosphorylated recombinant cPLA₂ to the nuclear envelope in cells reversibly permeabilized with ß-escin (B,C). (A) Recombinant cPLA₂ unphosphorylated (UP), phosphorylated (P) by purified CaMKII or after dephosphorylation (DP) by alkaline phosphatase (0.5 U ml^-1) was separated on 10% SDS-PAGE and analysed for gel shift as described in Materials and Methods. (B) VSMCs were reversibly permeabilized with ß-escin to introduce cPLA₂ (0.5 µg) that was unphosphorylated, phosphorylated by CaMKII or dephosphorylated by AP (0.5 Unit ml^-1) and detected by immunofluorescence staining. Phosphorylated, but not unphosphorylated or dephosphorylated, cPLA₂ translocated to the nuclear envelope (representative of three experiments). In nonpermeabilized cells, unphosphorylated, phosphorylated and dephosphorylated cPLA₂ did not translocate to the nuclear envelope. (C) Density of cPLA₂ fluorescence around the nuclear envelope (n=5). ^*Value significantly different from the corresponding value obtained with unphosphorylated (UP) cPLA₂ in permeabilized cells (P<0.05). (D) Unphosphorylated, phosphorylated and dephosphorylated cPLA₂ (0.5 µg) conjugated with fluorescence-tagged Alexa 488 was introduced in VSMCs reversibly permeabilized with ß-escin. The figure shows a representative of three experiments. Phosphorylated, but not unphosphorylated or dephosphorylated, cPLA₂ accumulated around the nuclear envelope.

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