Azido homoalanine

All cell lines used were obtained from ATCC (Manassas, VA) and have since been validated (Promega). DU145 (prostate carcinoma), H1993 (lung carcinoma), AU565, and HCC1806 (breast carcinoma) cells were maintained in RPMI 1640 media (Cellgro, Herndon, VA), MDA-MB-231 (breast carcinoma) cells were maintained in DMEM (Cellgro), and HEK293 cells in EMEM (Cellgro); each containing 10% fetal bovine serum (FBS) (Gemini, West Sacramento, CA). Cells were kept at 37°C with 5.0% CO₂. Monensin and brefeldin A were obtained from Biolegend (San Diego, CA), Y27637 from Stem Cell Technology (Vancouver, BC), C75, 2-hydroxymyristic acid, and 17-Octadecynoic Acid from Caymen Chemicals (Ann Arbor, MI), cycloheximide, recombinant EGF, geranylgeranyltransferase inhibitor, farnesyltransferase inhibitor, vinblastine, latrunculin, dansylcadaverine, ethylisopropyl amiloride, methyl-β-cyclodextrin, nystatin, and 2-bromopalmitate were obtained from Sigma-Aldrich (St. Louis, MO). Click-iT® reaction buffers, azido-homoalanine, azide-palmitate, and methionine-free RPMI, were obtained from Invitrogen (Carlsbad, CA). Recombinant HGF was obtained from EMD Millipore (Darmstadt, Germany). Streptavidin Sepharose High Performance beads were obtained from GE Healthcare (Pittsburgh, PA).

The AHA incorporation assay for estimating global translation was performed as previously described [19] . Briefly, cells were treated with IL-6 or pp242 in methionine-free media (Invitrogen), followed by a 30 minute exposure to 100 uM AHA (Azidohomoalanine, (Invitrogen)), a methionine analog to pulse label newly synthesized proteins. After cell lysis, incorporated AHA was detected using the TAMRA Click-iT protein Analysis Detection Kit (Invitrogen). Ten ug total protein was loaded per well. Following detection of newly synthesized protein, gels were also immunoblotted for GAPDH expression. TAMRA fluorescence (newly synthesized protein) was detected using a FujiFilm LAS-4000 system and analyzed using Multi Gauge V3.0 software. Newly synthesized protein levels were normalized to the GAPDH Western blot intensities.