Figure 4.

Oz-apraA induces Hsp90 client protein degradation through lysosomal pathway. (A) Lysosomal inhibitor abrogates oz-apraA–induced Hsp90 client protein degradation. A549 cells (left) and MDA-MB-453 cells (right) were pretreated with the indicated compounds for 2 h followed by treatment of 50 nM oz-apraA for 24 h. Cell lysates were separated by SDS-PAGE. The Western blot was probed with anti-EGFR, anti-RIP1, and anti-ErbB2 antibodies. Anti–α-tubulin was used as a loading control. (B) Immunolocalization of EGFR and lysosomal marker LAMP-1 in A549 cells before and after treatment with oz-apraA in the presence of 20 mM NH₄Cl. A549 cells were transiently transfected with LAMP-1–RFP expression plasmid, and after a 24-h transfection, cells were treated with or without 50 nM oz-apraA and 20 mM NH₄Cl for 6 h, fixed, permeabilized, and stained with anti-EGFR rabbit polyclonal antibody. Arrows indicate the colocalization of EGFR and LAMP-1. Bar, 15 µm.