Stain free page gels

Whole cell fractions were obtained as previously described.23 Protein concentrations were measured with the DC protein assay (Bio‐Rad Laboratories, Hercules, CA). Equal amounts of protein were loaded into lanes of CriterionTGX (Tris‐Glycine eXtended) Stain‐Free PAGE gels (BioRad). The gels were electrophoresed and activated using a ChemiDoc MP Visualization System (BioRad). The protein was then transferred to a polyvinylidene difluoride membrane. The membranes were then imaged using a ChemiDoc MP Visualization System to obtain an assessment of proper transfer and to obtain total protein loads. The membranes were then blocked and probed with primary antibodies overnight at 4°C. Immunoblots were next processed with secondary antibodies (Cell Signaling) for 1 hour at room temperature. Immunoblots were then probed with a Super Signal West Dura kit (Thermo Fisher Scientific) to visualize signal, followed by visualization using a ChemiDoc MP Visualization System (BioRad). Data were analyzed using Image Lab (BioRad). The total protein images were used as loading controls. For each protein of interest, the portion of the protein load image corresponding to the molecular weight of the protein of interest was used as the loading control.23

Whole cell fractions were obtained, as previously described.22 Protein concentrations were measured with the DC protein assay (Bio‐Rad Laboratories, Hercules, CA). Equal amounts of protein were loaded into lanes of Criterion TGX (Tris‐Glycine eXtended) Stain‐Free PAGE gels (BioRad). The gels were electrophoresed and activated using a ChemiDoc MP Visualization System (BioRad). The protein was then transferred to a polyvinylidene difluoride membrane. The membranes were then imaged using a ChemiDoc MP Visualization System to obtain an assessment of proper transfer and to obtain total protein loads. The membranes were then blocked and probed with primary antibodies overnight at 4°C. Immunoblots were next processed with secondary antibodies (Cell Signaling) for 1 hour at room temperature. Immunoblots were then probed with a Super Signal West Dura kit (Thermo Fisher Scientific) to visualize signal, followed by visualization using a ChemiDoc MP Visualization System (BioRad). Data were analyzed using Image Lab (BioRad). The total protein images were used as loading controls. For each protein of interest, the portion of the protein load image corresponding to the molecular weight of the protein of interest was used as the loading control.23