Skim milk

U251MG cells (10⁵ cells/1000 µL/well) were cultured in DMEM/F12 culturing condition (serum-free medium as recently described [14 (link)]) using a 24-well plate (Corning) for 72 h. U251MG Cells were lysed in ice-cold lysis buffer containing phosphatase inhibitor (CST). Whole proteins from cultured cells were extracted using cell lysis buffer following the manufacturer’s instructions (CST). Proteins were separated by SDS-PAGE in a 10% resolving gel and electro-transferred onto polyvinylidene difluoride membranes (Merck). Membranes were blocked in 3% skim milk (GE Healthcare, Minato-ku, Tokyo, Japan) in TBS-T at ambient temperature for 1 h with agitation, and incubated with the following primary antibodies overnight at 4 °C: rabbit polyclonal anti-RPS6 antibody (1:1000), mouse polyclonal anti-β-actin antibody (1:1000), rabbit polyclonal anti-STAT3 (1:1000), rabbit polyclonal anti-p-STAT3 (Tyr705) (1:1000), rabbit polyclonal anti-SOX2 (1:1000), and mouse monoclonal anti-Nestin (1:1000) antibodies. After three washes, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (rabbit: GE Healthcare, mouse: GE Healthcare) with 3% skim milk for 1 h. Finally, immunoreactive protein bands were visualized by using ECL select detection reagents (GE Healthcare) and captured using the LAS4000EPUVmini (GE Healthcare).

Cell lysates or culture medium was subjected to 15% gel SDS-PAGE or gradient gel native-PAGE, respectively, and then transferred to polyvinylidene difluoride (PVDF) membranes. After blocking the membrane with 2% skim milk (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TTBS) for 1 h, the membranes were treated with a primary antibody in the blocking solution at 4°C overnight. Furthermore, after washing the membrane with TTBS, the membrane was treated with a secondary antibody in TTBS containing 0.5% skim milk at RT for 2 h. The protein bands were detected using ECL-plus western blotting detection reagent (GE Healthcare UK Ltd., Buckinghamshire, UK) by ImageQuant LAS500 (GE Healthcare). Densitometric analysis was performed using ImageJ software.