Figure 7. Irradiation induces the release of Hb-EGF from isolated plasma membranes.

(A) Plasma membranes of serum-starved HeLa cells were isolated as described in the Experimental section. Membranes (5 μl of net membranes dissolved in 100 μl of PBS in each condition) were incubated with NAC (2.5 mM) or GM-6001 (0.5 μM), or were left untreated (None) for 15 min. Membranes were then irradiated at 875 MHz with an intensity of 0.300 mW/cm² for the indicated times and subjected to Western blot analysis with the anti-(Hb-EGF) antibody (α Hb-EGF; top panel). The membranes, as well as total cell extract (Total), were also analysed with the anti-EGFR (αEGFR) and -(total ERK) (αERK) antibodies (middle and bottom panels respectively). (B) Isolated plasma membranes from serum-starved HeLa cells, prepared as above, were incubated with 100 and 200 μM H₂O₂ for the indicated times. The amount of Hb-EGF and EGFR was determined with the appropriate antibodies. (C) Quantification of Hb-EGF secretion. Values are means±S.E.M. for three separate experiments. GM, GM-6001; Rad, radiation.