Tristetraprolin

Total protein extracts were obtained by lysing the cells in RIPA buffer (50 mM TRIS-HCl pH 7.4, 150 mM NaCl, 1% NP40, 1 mM sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM EDTA) with addition of Protease Inhibitor Cocktail 1x (Roche Applied Science, Penzberg, Germany). Cells were resuspended and centrifuged in the buffer and subsequently quantified by Bradford assay. A fixed amount of protein for each sample was loaded on SDS-Polyacrylamide gels for each sample and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in TBS + 0.1% Tween, and then immunoblotted overnight at 4 °C with the following primary antibodies: Tristetraprolin (#71632, Cell Signalling Technologies, Danvers, MA, USA); TWIST1 (#46702, Cell Signalling Technologies, Danvers, MA, USA); SLUG (#9585, Cell Signalling Technologies, Danvers, MA, USA); SNAI1 (#3895, Cell Signalling Technologies, Danvers, MA, USA). One-hour incubation at room temperature was enough for the primary antibody against CTNNB1 (BD Transduction Laboratories 610154, South San Francisco, California, USA). The nitrocellulose membranes were then incubated with secondary antibodies: either Goat anti-rabbit IgG-HRP or Goat anti-mouse IgG-HRP (Santa Cruz Biotechnology sc-2004 and sc-2005, Santa Cruz, CA, USA). Chemiluminescence was detected with ChemiDoc (BIO-RAD, Hercules, California, USA).

Cells were lysed in RIPA buffer supplemented with 2μg/mL aprotinin, 2μg/mL leupeptin, 0.7μg/mL pepstatin, 20mM sodium fluoride, 1mM sodium orthovanadate, 1mM dithiothreitol, 10mM beta-glycerophosphate, and 10mM sodium pyrophosphate. Following protein quantification using Bradford reagent, western blots were performed using standard procedures. The following commercially available antibodies were used for immunoblotting: tristetraprolin (Cell Signaling Technology 71632, 1:500), BRF1/2 (Cell Signaling Technology 2119, 1:1000), ERK (Cell Signaling Technology 9102, 1:1000), phospho-Thr202/Tyr204 ERK (Cell Signaling Technology 4370, 1:1000), S6 kinase (Cell Signaling Technology 2708, 1:1000), phospho-T389 S6 kinase (Cell Signaling Technology 9234, 1:500), c-Myc (Cell Signaling Technology 18583, 1:1000), GAPDH (Cell Signaling Technology 5174, 1:1000), β-actin (Cell Signaling Technology 3700, 1:1000) and α-tubulin (Sigma T6074, 1:10,000).