Rabbit α pttg1

Cells were fixed with 3.7% formaldehyde for 15 min at RT, permeabilized with 0.05% Triton X-100 in PBS, and blocked with 5% bovine serum albumin (BSA). Cells were incubated with primary antibodies (rabbit α-PTTG1, Abcam; mouse α-ZEB1, Santa Cruz) and then incubated with goat Alexa Fluo-488 anti-rabbit IgG and/or goat Alexa Fluo-594 anti-mouse IgG (Molecular Probes, Eugene, OR, USA).

For Western blot, cells were lyzed in RIPA buffer (50 mM Tris–Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% Na deoxycholate, 0.1% SDS, 1 mM EDTA). Proteins were resolved by SDS-PAGE and then transferred onto PVDF membranes (Millipore). All buffers contained a cocktail of protease inhibitors (Boehringer, Ingelheim am Rhein, Germany). Membranes were developed using the enhanced chemiluminescence (ECL westar, Cynagen, Bologna, Italy). Bands were analyzed by chemiluminescence imaging system, Alliance 2.7 (UVITEC, Cambridge, UK) and quantified by the software Alliance V_1607.
The following primary antibodies were used: rabbit α-PTTG1 (Abcam), mouse α-MMP-2 (Thermo Fisher, Waltham, MA, USA), mouse α-HA (BioLegend, San Diego, CA, USA), mouse α-tubulin (Sigma, St. Louis, MO, USA), mouse α-actin monoclonal antibody C-40 (Sigma), and rabbit α-Sp1 (Santa Cruz, Santa Cruz, CA, USA).

Cells are fixed with 3.7% formaldehyde for 15 min at room temperature (RT), permeabilized with 0.05 Triton X-100 in PBS, and blocked with 5% bovine serum albumin (BSA). Cells were incubated with primary antibodies (rabbit α-PTTG1, Abcam, Cambridge, UK; mouse α-PBF, Novus Biologicals, Centennial, CO, USA) and then incubated with goat Alexa Fluo-488 anti-rabbit IgG and/or goat Alexa Fluo-594 anti-mouse IgG (Molecular Probes, Thermo Fisher, Waltham, MA, USA). DNA was stained with Prolong Gold DAPI (Molecular Probes).