β actin sc 47778 antibody

The whole-cell proteins were extracted by lysing cells in the RIPA buffer containing protease inhibitors and phosphatase inhibitors (Thermo Fisher) for 30 min on ice. Nuclear proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech). Briefly, cells were detached with 0.25% trypsin-EDTA and collected into a 1.5-ml Eppendorf tube. Cells were centrifuged at 250 r/min for 5 min and the cell pellet was incubated in 1× pre-extraction buffer (50 μl of buffer for 1×10⁶ cells) on ice for 10 min. Cells were vortexed for 10 s and centrifuged at 12,000 r/min for 1 min. After discarding the supernatant, the nuclear pellet was incubated in 40 μl of 1× extraction buffer on ice for 15 min. The suspension was centrifuged at 14,000 r/min for 10 min at 4°C, and the supernatant was collected for Immunoblotting. The β-actin (sc-47,778) antibody was purchased from Santa Cruz Biotechnology. The ETV6 antibody (PA5-79,224) was purchased from Thermo Fisher Scientific. The phospho-IKKα/β (Ser176/180) antibody (2694), and IKKβ antibody (8943), IκBα antibody (4814), NF-κB p65 antibody (8242), phospho-NF-κB p65 (Ser536) (E1Z1T), and Lamin B2 antibody (13,823) were purchased from Cell Signaling Technology. The results were recorded on an Azure c300 Gel Imaging System (Azure Biosystems) and quantified by the ImageJ.