Protein Quantification by SDS-PAGE and Western Blot

SDS-PAGE was performed on precast 10.5–14% Criterion™ Tris-Tricine gels, 10.5–14% or 4–10.5% Tris–HCl gels (Bio-Rad). Protein levels were normalized using 10–100 μg of protein per sample (depending on the targeted protein). The samples were resuspended with 4 × Tricine loading buffer and boiled for 5 min before loading. Proteins were transferred onto 0.2-μm nitrocellulose membrane (Bio-Rad) following electrophoresis. Membranes were blocked in 5% BSA (Sigma; St. Louis, MO, USA) in TBSTw for 1–2 h at room temperature and probed with the appropriate antisera/antibodies diluted in 5% BSA in TBSTw. Primary antibodies were probed with secondary antibodies conjugated with infrared dyes (LI-COR Biosciences; Lincoln, NE, USA). Densitometry analyses were performed using the Odyssey software (LI-COR Biosciences). Normalization was performed against GAPDH. Quantification by software analysis was performed as described previously [5 (link), 56 (link), 59 (link)–61 (link), 103 (link)].