Anti her2 29d8

Each cell line was seeded at about 2 × 10⁵cells per well in 6-well plates and then transfected with miRNA oligos as mentioned above when cells were be 50-70% confluent. After 48 h, cells were lysed with radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime). Then sample buffer containing suspended proteins was centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. A total of 30 μg of each protein sample was loaded in each well and separated with 10% SDS-PAGE. The proteins in the gel were electroblotted onto PVDF membranes (Millipore, Billerica, MA, USA) with wet blotting. Membranes were incubated in blocking buffer (1× Tris-buffered saline [TBS], 0.1% Tween-20, and 5% w/v dry nonfat milk) for 1 h at room temperature, and then membranes were incubated with anti-p53 DO-1 (1:300, Santa Cruz, Dallas, Tex, USA), or anti-Her2 29D8 (1:300, Cell Signaling Technology, Beverly, MA, USA), or anti-β-actin (sc-47778, 1:1000, Santa Cruz) for overnight at 4°C, followed by incubation with an appropriate peroxidase-conjugated secondary antibody at room temperature for 1 h. Reactive bands were detected by enhanced chemiluminescence (Millipore).