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First published online 23 April 2003
doi: 10.1242/jcs.00446

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Association of cPLA₂- and COX-1 with the Golgi apparatus of A549 human lung epithelial cells

School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK

View larger version (25K): [in a new window]   Fig. 1. Distribution of cPLA2- in resting and A23817-stimulated A549 cells. (A) cPLA2- was detected in A549 cell lysates (20 µg protein) using a goat polyclonal antibody (lane 1). Also shown is the control corresponding to goat polyclonal antibody pre-incubated with blocking peptide (lane 2). (B) Cells were grown on coverslips and incubated with buffer alone (control) or stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. Cells were then fixed and permeabilized, and cPLA2- was detected using immunofluorescence microscopy. Bar, 10 µm.

View larger version (44K): [in a new window]   Fig. 2. Distribution of cPLA2- compared with Golgi and ER markers. Cells were stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. Cells were then incubated with goat polyclonal anti-cPLA2- followed by AlexaFluor488-conjugated anti-goat antibody with either rhodamine-conjugated WGA (A) or rhodamine-conjugated ConA (B). Cells were visualized using immunofluorescence microscopy. Bar, 10 µm.

View larger version (43K): [in a new window]   Fig. 3. Distribution of cPLA2- compared with markers for the ER-Golgi intermediate compartment and the cis-Golgi cisternae. Cells were stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. Cells were then incubated with goat polyclonal anti-cPLA2-, with either mouse monoclonal anti-ERGIC53 (A) or mouse monoclonal anti-ß-COP (B) followed by anti-goat AlexaFluor488 and anti-mouse AlexaFluor594 secondary antibodies. Cells were visualized using immunofluorescence microscopy. Bar, 10 µm.

View larger version (40K): [in a new window]   Fig. 4. Distribution of cPLA2- compared with markers for the trans-Golgi stack and the TGN. Cells were stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. Cells were then incubated with goat polyclonal anti-cPLA2-, with either mouse monoclonal anti-GalT (A) or mouse monoclonal anti-TGN46 (B) followed by anti-goat AlexaFluor488 and anti-mouse AlexaFluor594 secondary antibodies. Bar, 10 µm.

View larger version (14K): [in a new window]   Fig. 5. Analysis of the degree of cPLA2- overlap with markers specific for Golgi subcompartments. The percentage overlap was calculated as described in the Materials and Methods. The graph represents results obtained from four independent experiments (±s.d.).

View larger version (41K): [in a new window]   Fig. 6. Effects of BFA treatment on the localization of cPLA2-. Cells were stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. BFA (10 µg/ml) was then added to the cells for 30 minutes and the cells were fixed and permeabilized. cPLA2- was then detected by immunofluorescence microscopy. Bar, 10 µm.

View larger version (45K): [in a new window]   Fig. 7. (A) Distribution of cPLA2- compared with COX-1 and COX-2. Cells were grown on coverslips and stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. Cells were then incubated with goat polyclonal anti-cPLA2-, with either mouse monoclonal COX-1 (A) or mouse monoclonal COX-2 (B) followed by anti-goat AlexaFluor488 and anti-mouse AlexaFluor594 secondary antibodies. Bar, 10 µm. (C) Quantification of the degree of cPLA2- overlap with COX-1 and COX-2. Percentage overlap was calculated as described in the Materials and Methods. The graph represents results obtained from four independent experiments (±s.d.).