Immobilon 0.45 μm

Western blot analyses were performed on cell lysates prepared from MDA-MB-231 and Hs578T cell lines as described previously [12 (link)]. Briefly, triplicate cell cultures were first washed with phosphate buffered saline (PBS, Invitrogen) and then lysed by mixing 1:1 with 2× sodium dodecyl sulphate sample buffer (100 mM Tris–HCl, pH = 6.8, 200 mM DTT, 4% SDS, 20% glycerol and 0.002% bromoplenol blue). Cell lysates were separated by 10% SDS-PAGE. Proteins were transferred to PVDF membranes (Immobilon 0.45 μm, Millipore, USA) and immersed in a blocking solution containing 5% non-fat milk and 0.1% Tween-20 for 1 h. The membranes were washed and incubated with primary antibodies (rabbit polyclonal anti-alpha-tubulin (abcam) at 1:1000 dilution, rabbit polyclonal anti-KIAA1199 (Sigma-Aldrich) at 1:100 dilution, rabbit ployclonal anti-KIAA1199 antibody (Protein Tech Group, Chicago, IL) at 1:800 dilution or rabbit anti-Caspase-3 (8G10) monoclonal antibody (Cell Signaling) at 1:1000 dilution) for 2 h and then with secondary antibodies for 1 h at room temperature. After washing the resulting bands were visualized using the standard ECL procedure, quantified by densitometry and normalized to the corresponding α-tubulin bands.

Cells were collected and lysed in RIPA lysis buffer (Solarbio). A total of 10 μL of each protein extract were added into 6%–15% SDS-PAGE gels and transferred to PVDF membrane (Immobilon 0.45 μm, Millipore, USA). The resulting blots were immersed in the first antibodies diluted with 5% non-fat milk overnight at 4°C, including PLCE1 (sc-28404, 1:200), Bax (ab32503, 1:1000), cleaved-PARP (ab32064, 1:5000), Snail (13099-1-AP, 1:250), Slug (12129-1-AP, 1:250), caspase-3 (19677-1-AP, 1:250), caspase-7 (BA0688-1, 1:125), Bcl-2 (AB112, 1:1000), and β-actin (Zhongshanjinqiao 140411, 1:1000); and then treated with a secondary antibody for 2 h at room temperature. Finally, the resulting bands were exposed using the standard ECL procedure. Protein was normalized with β-actin.