Total protein lysate preparation from cells and organ tissues for pull-down or immunoblot are described (75 (link), 76 (link)). Protein concentrations were determined by the bicinchoninic acid (BCA) Protein Assay Kit (Pierce), and 20 μg from each sample was separated by NuPAGE bis-tris 4 to 12% gradient SDS–polyacrylamide gel electrophoresis (PAGE) (Invitrogen) and transferred onto polyvinylidene difluoride membrane (Bio-Rad), probed with the primary antibodies, with horseradish peroxidase (HRP)–conjugated mouse or rabbit secondary antibodies (Cell Signaling Technology) and enhanced chemiluminescence (Thermo Fisher Scientific; SuperSignal West Femto Maximum Sensitivity Substrate).
Immunoblots were imaged by ProteinSimple FluorChem M using Auto Exposure feature that takes successively longer exposures of the membrane until an optimum exposure is achieved. Images are acquired in standard resolution, using 4 × 4 pixel binning (832 × 626–pixel images). Multichannel imaging captures fluorescent markers immediately adding chemiluminescent. The system includes three tools to enhance visibility of the results: brightness control (using nonlinear gamma adjustment), exposures view, and color inversion. Densitometric analysis of protein quantitation was determined by ImageJ software v0.5.5 (NIH;http://imagej.nih.gov/ij , Java 1.8.0- internal).
Immunoblots were imaged by ProteinSimple FluorChem M using Auto Exposure feature that takes successively longer exposures of the membrane until an optimum exposure is achieved. Images are acquired in standard resolution, using 4 × 4 pixel binning (832 × 626–pixel images). Multichannel imaging captures fluorescent markers immediately adding chemiluminescent. The system includes three tools to enhance visibility of the results: brightness control (using nonlinear gamma adjustment), exposures view, and color inversion. Densitometric analysis of protein quantitation was determined by ImageJ software v0.5.5 (NIH;