Anti mtdh

Total protein was purified from cultured cells with Radio-Immunoprecipitation Assay (RIPA) Buffer (SIGMA-ALDRICH) and preserved at-80°C. Thirty micrograms of protein were loaded onto a 7.5-15% XV PANTERA Gel (DRC, Tama, Tokyo, Japan), and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were blocked at room temperature for 60 minutes in 5% milk in 0.1% Tris Buffed Saline with Tween-20 (TBS-T). Membranes were incubated with anti-MTDH (Invitrogen; 1:1000), anti-E-cadherin (Cat#sc-7870, Santa Cruz; 1:1000), anti-Twist1 (Santa Cruz; 1:300), and anti-β-actin (Cat#13E5, Cell Signaling, Danvers MA, USA; 1:2000) antibodies overnight at 4°C, washed three times for 5 minutes each with TBS-T, and incubated with secondary antibodies (anti-rabbit IgG horseradish peroxidase; Santa Cruz Biotechnology; 1:3000). The membranes were developed using ImageQuant LAS-4000UV mini Mac (General Electoric Company, Fairfield, CT, USA) after immersion in the detection reagent. Western blots were quantified by densitometry analysis and normalized to β-actin using the Image J software.

Cells were lysed and protein concentration was determined using the BCA Assay Kit (Thermo Scientific). Protein samples were separated by SDS-PAGE and electrotransferred onto PVDF membrane (Millipore). After blocking with 5% non-fat milk, the membrane was incubated overnight at 4°C with the primary antibody and then with horseradish peroxydase-coupled secondary antibody. Signal was detected with enhanced chemiluminescence (ECL) (PerkinElmer) by ImageQuant LAS 4000 (GE Healthcare Life Sciences). Antibodies used include anti-MTDH (Invitrogen), anti-P53 (DAKO), anti-β-actin (Sigma-Aldrich). Other antibodies used in this study were from Cell signaling Technology.