Figure 7. K5 interacts with and alters RTK localization.

Equal cell numbers of the indicated THP-1 lines were fixed with paraformaldehyde (PFA) and (A) stained without permeabilization to determine surface expression or (B) permeabilized with saponin prior to staining to determine total expression of Flt-4, PDGFR-ß, Flt-3 and EGFR by flow cytometry. Data are representative of three independent experiments. (B, Inset) The relative ratio of surface versus total RTKs was determined for vector- and K5 WT-expressing THP-1 cells. (C) 293T cells were co-transfected with expression constructs for EGFR, PDGFR-ß, or Flt-4 and the indicated GST expression constructs. After two days, lysates were subjected to GST pull-down using glutathione-sepharose beads. Purified proteins and whole cell lysates (WCL) were subjected to western blot (WB) using anti-EGFR, -Flt-4 or -PDGFR-ß antibodies, followed by re-probing with anti-GST antibodies. Arrows indicate GST or GST-K5 WT and mutant specific bands. Data are representative of three independent experiments.