Ecl2 western blotting substrate kit

Whole cell extracts were created by lysing cell pellets with RIPA buffer (Boston BioProducts, Ashland, MA) for 20 min on ice, and then lysates were cleared by centrifugation for 10 minutes at 15,000 rpm at 4°C. Samples were loaded onto NuPage 4–12% Bis-Tris gradient gels (Novex, Life Technologies, Grand Island, NY) and separated by electrophoresis in MOPS-SDS running buffer. Proteins were transferred to nitrocellulose membranes via the iBlot dry transfer system (Invitrogen, Life Technologies, Grand Island, NY). Blots were blocked for 1 hour at room temperature in 5% nonfat milk in PBS-Tween-20 (Westnet Inc., Canton, MA) and incubated in appropriate primary antibodies diluted in blocking buffer overnight at 4°C (Supplemental Table S1). Blots were then incubated in HRP-linked secondary antibody (GE Healthcare, Piscataway, NJ) at 1:4,000 dilution in blocking buffer. Proteins were detected using the ECL2 western blotting substrate kit (Thermo Fisher Scientific, Waltham, MA) and imaged with a FluorChem HD2 imager (Cell Biosciences, Santa Clara, CA). After initial development, membranes were re-probed with antibodies to β-actin (Sigma-Aldrich, St. Louis, MO) as a loading control.

Whole cell extracts were created by lysing cell pellets with RIPA buffer (Boston BioProducts) for 20 minutes on ice, and then cleared lysates were quantified by Bradford assay. Samples were loaded onto NuPAGE 4–12% Bis-Tris gradient gels (Novex, Invitrogen) and separated by electrophoresis in MOPS-SDS running buffer. Proteins were transferred to nitrocellulose membranes via the iBlot dry transfer system (Invitrogen). Blots were blocked for 1 hour at room temperature in 5% nonfat milk in PBS-Tween-20 (Westnet Inc.) and incubated with a rabbit polyclonal antibody against PAX8 (Proteintech, 10336-1-AP) diluted 1:1,000 in blocking buffer overnight at 4°C. Blots were then incubated in HRP-linked secondary antibody (GE Healthcare) at 1:4,000 dilution in blocking buffer. Proteins were detected using the ECL2 Western blotting substrate kit (Thermo Fisher Scientific) and imaged with a FluorChem HD2 imager (Cell Biosciences). After initial development, membranes were reprobed with a mouse monoclonal antibody to β-actin (Sigma-Aldrich, A2228), diluted 1:2,500 in blocking buffer, as a loading control.