Multi gauge v3

Proteins of cells and tissues were extracted with the lysis buffer (the composition: 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, and 1% NP-40) and protein levels were determined by a Bradford protein Assay Reagent Kit (Bio-Rad, Hercules, CA, USA). The equal amounts of each protein samples were loaded in the 8% SDS polyacrylamide gel electrophoresis (SDS-PAGE). Immun-Blot^® polyvinylidene difluoride membranes (Bio-Rad) were used to transfer proteins from SDS-PAGE. After blocking with specific blocking buffer (Goal Bio, Taipei, Taiwan) for 2 min at room temperature, membranes were probed with primary HER2 antibody (1:2000) (Sigma-Aldrich) at 4 °C overnight. After washing membranes under standard washing procedure, membranes were probed with secondary antibody (dilution rate: 1:3000) (Sigma-Aldrich) at 4 °C for 1 h. The immunoreactive complexes were reacted with enhance chemiluminescence (Clarity™, Bio-Rad) and detected by using a LAS-4000 mini luminescent image analyzer (GE Healthcare; Uppsala, Sweden). Band densitometry was quantified by Multi Gauge v3.2 software (GE Healthcare).

For the western blot analyses, RIPA buffer containing protease inhibitors and phospha tase inhibitors (Roche, Basel, Switzerland) was used to prepare whole-cell lysates. Briefly, equal amounts of lysate were separated by SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membranes (Millipore,Massachusetts, USA). After blocking with 5% bovine serum albumin, the membranes were probed with anti-FGF9 or CCND2 and anti-GAPDH (ab71395, ab226972, ab9485, Abcam, Cambridge, UK), followed by incubation with a horseradish peroxidase–conjugated secondary antibody [goat-anti-mouse IgG (1:2000) and goat-anti-rabbit IgG (1:3000)]. The proteins were visualized using Image Reader LAS-4000 (Fujifilm) and analyzed with Multi Gauge V3.2 software (GE Healthcare Life Sciences, USA).

The samples were loaded in a 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE), and then the proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad; Hercules, CA, USA). After blocking 30 min at 4 °C (blocking reagent, Goal Bio, Taipei, Taiwan), the membranes were then incubated with primary antibodies against CD166 (1:2000) (Sigma-Aldrich), Nanog (1:1000), c-Myc (1:1000), OCT4 (1:2000), and Survivin (1:2000) (Cell signaling technology; Danvers, MA, USA) at 4 °C overnight. After washing procedure, membranes were incubated with secondary antibody (1:3000) (Sigma-Aldrich) at 4 °C for 1 h. Finally, the membranes were covered with enhance chemiluminescence substrate (Thermo Fisher Scientific) for 1 min and analyzed by using a luminescent image analyzer (LAS-4000 mini; GE Healthcare, Uppsala, Sweden). Band densitometry was quantified by Multi Gauge v3.2 software (GE Healthcare).