Goat anti mouse igg hrp linked antibody

Whole-cell lysates were prepared with a proteinase inhibitor cocktail and phosphatase inhibitor cocktail (Roche, Alameda, CA, USA). Protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Equal amounts of proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Danvers, MA, USA). Membranes were blocked with 5% non-fat dry milk containing 0.1% Triton X-100 at room temperature and blotted with primary antibodies overnight at 4 °C. Signals were visualized by chemiluminescence (Thermo Fisher Scientific, Grand Island, NY, USA). Goat anti-rabbit IgG HRP-linked antibody (1:5000) and goat anti-mouse IgG HRP-linked antibody (1:5000) were from Proteintech (Rosemont, IL, USA).

Whole cell lysates of NPC cells were prepared with a proteinase and phosphatase inhibitor cocktail (Roche, CA, USA). Equal amounts of proteins were resolved to 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Danvers, MA, USA) and blocked with 5% nonfat dry milk in Tris-buffered saline, pH 7.5. Membranes were immuno-blotted overnight at 4 °C. Protein blots were probed with primary antibodies against HIPK2 (Abcam #ab108543), E-cadherin (Cell Signaling Technology #3195), N-cadherin (Cell Signaling Technology #13116), SPEN (Abcam #ab72266), c-JUN (Abcam #ab40766), phosphor-AKT (Cell Signaling Technology #4060), AKT (Cell Signaling Technology #4691), phosphor-PI3K (Cell Signaling Technology #17366), and PI3K (Cell Signaling Technology #4292). Goat anti-rabbit IgG HRP-linked antibody (1:10,000) and goat anti-mouse IgG HRP-linkedantibody (1:10,000) were from Proteintech (Rosemont, IL, USA). Signals were visualized by chemiluminescence (Bio-rad, Hercules, California) and quantitated using a Quantity One system (Bio-Rad, Hercules, CA, USA).