Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide
Figure 9
LOX-PP inhibits the migratory phenotype of lung cancer cells via interaction with c-Raf.
(A) H1299 cells were transfected with expression plasmids for GST or LOX-PP-GST (PP-GST) and tagged proteins purified on Glutathione-Sepharose 4B beads. Bound proteins were analyzed by immunoblotting for the indicated proteins. For estimation of the amounts of expressed proteins, 4% of each of the lysates was immunoblotted (Input). (B) Triton X-100 extracts of A549 were immunoprecipitated with the indicated antibodies. The precipitated proteins were analyzed by Western blotting for c-Raf and LOX-PP. As the band of precipitated LOX-PP migrated close to that of rabbit IgG light chain, Protein A-conjugated HRP was used as a secondary ‘antibody’ to detect immunoprecipitated LOX-PP. (C) GST (EV), GST-LOX-PP WT (G-PP-WT), or GST-LOX-PP ΔM3 (G-PP-ΔM3) were expressed in H1299 cells for 24 h and tagged proteins purified as in part A and subjected to Western blotting with the indicated antibodies. Input = 4% of the lysate. (D) Either GST (EV), GST-LOX-PP WT or GST-LOX-PP ΔM3 (Δ26–100) were expressed in H1299. After 24 h, cells were subjected to a migration assay in triplicate for 16 h. Cells that migrated to the lower side of the filter were stained and quantified by spectrometric determination at A570 nm. The average values from three independent experiments ± SD presented relative to the EV control, set to 1.0. P values were calculated from three independent experiments using Student's t test. *, P<0.01. (E) GST (EV) or GST-LOX-PP (PP) was co-transfected in H1299 cells in presence of a vector expressing a constitutively active MEK mutant (CA-MEK) or EV DNA (−) for 24 h. Cultures were subjected to a migration assay for 16 h, in triplicate, as in part D. P values of three independent experiments were calculated using Student's t test. *, P<0.01.