Lf ma30327

Cells and mammospheres were lysed in ice in a RIPA buffer (1% NP-40, 150 mM NaCl, 1% sodium deoxycholate, 0.1 % SDS, 25 mM Tris-HCl pH 7.6). The lysates were centrifuged and the cytoplasmic extract was collected in the resulting supernatant. Samples (20 μg/10 μL) were prepared from the cells and mammospheres. After electrophoresis on a 12% SDS-PAGE gel, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). The membrane was incubated in an Odyssey blocking buffer at room temperature for 1 h and then incubated overnight with primary antibodies. The antibodies were p65, LF-MA30327; GAPDH, LF-PA0018; Oct4, LF-MA30482 (AbFrontier, Seoul, Korea); LaminB, sc-365962; Nanog, sc-293121; Sox2, sc-365923 (Santa Cruz Biotechnology, Dallas, TX, USA); and c-Myc, 551101 (BD, San Jose, CA, USA). After the membranes were washed, they were incubated with IRDye 680RD and 800W secondary antibodies at room temperature for 1 h, and the signals were determined with an Odyssey CLx machine (Li-Cor, Lincoln, NE, USA).

We used a previously described method for western blotting [35 ]. Proteins samples (20 μg/well) of mammospheres treated with 6-methoxymellein were isolated and run on a 10% SDS-PAGE gel. The separated protein gels were transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Membranes were incubated at room temperature for 1 h in Odyssey blocking buffer in PBST (0.1% Tween 20). The membranes were incubated at 4 °C overnight with the following primary antibodies: NF-κB p65, LF-MA30327, NF-κB p50, sc-8414; Oct4, LF-MA30482 (AbFrontier, Seoul, Korea); c-Myc (Cell Signaling Technology, Denver, CO, USA); Sox-2, Lamin B and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). After the membranes were washed with 1x PBST, they were incubated with IRDye 800CW and 680RD-conjugated secondary antibodies for 1 h, and band densities were examined by using an ODYSSEY CLx (LI-COR, Lincoln, NE, USA).