Figure 7

Identification and verification of Photofrin-interacting proteins. (a) SDS-PAGE analysis of potential Photofrin-interacting proteins. A431 cell lysates (1 mg of protein) were incubated with Photofrin-coupled beads (P) or control beads (Ctrl), and washed with 1 M NaCl and PBS. The bead-bound proteins (Bound) and the unbound supernatants (Sup) were subjected to SDS-PAGE followed by silver staining. T, total cell lysates. (b) The proteomics workflow used to identify potential Photofrin-interacting proteins. See the Materials and Methods for details. (c) Samples were processed as described in (a), and the bead-bound proteins (Bound) were subjected to SDS-PAGE followed by Western blot analysis with anti-EGFR or anti-cathepsin D antibodies. T, total cell lysates. (d) A431 cells or recombinant EGFR (or cathepsin D) proteins were left untreated (Un) or treated with laser irradiation (L), Photofrin (P), or both (PDT), and the cell lysates or reaction products were subjected to Western blot analysis with anti-EGFR (left panel) or anti-cathepsin D (right panel) antibodies. (e) Recombinant EGFR was treated as described in (d) and then incubated with (+) or without (−) kinase assay buffer containing ATP.Mg²⁺at 30 °C for 10 min. The reaction products were subjected to Western blot analysis with anti-EGFR or anti-phosphotyrosine antibodies. (f) Recombinant cathepsin D was treated as described in (d), and the untreated/treated cathepsin D proteins were incubated with a reaction mixture containing 0.5 μg proinsulin at 37 °C for 10 or 30 min. The reaction products were analyzed by SDS-PAGE followed by silver staining. The arrow and arrowhead indicate proinsulin and its processed product, respectively.