Fig. 2. (A) Western blot analysis using a polyclonal rabbit anti-rat HB-EGF antibody and total cellular lysate from the cells used in the studies. (B) Release of soluble HB-EGF in the culture medium. Serum free conditioned medium from the control and HB-EGF-transfected cells was loaded onto pre-equilibrated heparin-sepharose columns, washed with 10 mM Tris-cl, pH 7.4 and 100 mM NaCl and eluted with 2 M NaCl. Equal amount of eluants was resolved on 12% SDS-PAGE and probed with anti-HB-EGF antibody. The observations are representative of data from two independent clones for each transfected molecule and three independent experiments. (C) A Paracrine assay to establish the lack of cleavage of proHB-EGF in cells expressing deletion mutant constructs of HB-EGF. Equal amounts of the eluants from the purified conditioned media of control and HB-EGF-transfected cells were added to the quiescent A431 cells for 5 minutes. Cells were washed with PBS and lysed in RIPA buffer. Equal amounts of protein were resolved on 6% SDS-PAGE and probed with anti-phospho-EGFR antibody. The observations are representative of data from two independent clones for each transfected molecule and at least three independent experiments. The same blot was stripped and probed with the anti-EGFR antibody to verify equal protein loading. (D) Effect of phorbol myristate acetate ester (PMA; 1 µM) on the cleavage of proHB-EGF and deletion mutant constructs of HB-EGF. Quiescent cells were treated with 1 µM of PMA for 30 minutes. Cells were washed with 2 M NaCl to remove any soluble HB-EGF bound to the cell surface. Equal amount of protein was immunoprecipitated with anti-FLAG (M2) antibody. Samples were resolved on 10% Tricine gel and probed with anti-FLAG antibody. The (+) indicates cells treated with PMA and (–) indicates nontreated cells. The full-length HB-EGF and residual transmembrane/cytoplasmic domain remaining after cleavage of proHB-EGF are indicated by arrows and arrowheads, respectively. The observations are representative of data from two independent clones for each transfected molecule and at least three independent experiments. (E) EGF receptor expression in confluent MDCK control and HB-EGF-transfected cells. The basal EGF receptor tyrosine phosphorylation in quiescent MDCK control and HB-EGF-transfected cells. The observations are representative of data from two independent clones for each transfected molecule and five independent experiments.