(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.
Fig. 6. (A) Calcium release is stimulated by activation of integrin ß1. FLUO3/AM-labelled serum-starved BE cells were placed in suspension and stimulated with either the activating integrin ß1 antibody (TS2/16 10 µg/ml; top panel) or Mn2+ (5 µM; middle panel). Changes in the intracellular calcium concentration were then measured using a fluorimeter (ex. 490 nm, em. 525 nm). Inhibitors U73122 (2 µM) or PP2 (10 µM) were included in these incubations where indicated. Representative traces are shown, and quantified intracellular calcium concentration, which was analysed from three independent experiments is reported with standard deviations: basal/unstimulated calcium 100 nM; TS2/16 stimulation: control 297.6±12.6 nM, +PP2 109.2±16.9 nM, +U73122 99.5±5.4 nM. Mn2+ stimulation: control 326.5±13.5 nM, +PP2 100.3±13.1 nM, +U73122 90.5±17.4 nM. Fura2/AM labelled BE cells were also plated on Matrigel and calcium release anlaysed by dual-colour real-time fluorescent microscopy at excitation wavelengths of 380 nm (green: low/basal intracellular calcium) and 340 nm (red: high/released intracellular calcium) (emission 510 nm). Sample images at various time points were taken from the videos and are displayed (lower panel). (B) PKC phosphorylation status is unaffected by stimulation or inhibition of integrin ß1. Cells plated on Matrigel were either left untreated (control) or treated with the integrin ß1 blocking antibody 4B4 (10 µg/ml; anti-ß1), PP3 or PP2 for 8 hours, recovered from the matrix and processed for western blotting using antibodies to phosphorylated or unphosphorylated forms of PKC as indicated (left panels). Serum-starved BE cells on plastic were left unstimulated (control) or stimulated with either the activating integrin ß1 antibody (TS2/16 10 µg/ml) or Mn2+ (5 µM) for 30 minutes, lysed and processed for western blotting as when analysed on Matrigel (middle panel). As a control for the antibody specificity, A431 cells grown on plastic were either left unstimulated, stimulated with EGF (100 ng/ml) or stimulated with EGF (100 ng/ml) in the presence of AG1478 (10 µM) inhibitor (right panel).
