Luminol enhanced chemiluminescence ecl hrp substrate

Western blotting was performed as described previously (Lin et al., 2014a (link),b (link)). Protein content was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, 23228). Equal amounts of total protein (30 µg tissue homogenate per lane) were subjected to 4-12% NuPAGE gel for electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 3% nonfat milk and incubated with primary antibody overnight at 4°C. Blots were washed 3×10 min in TBST, then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology) for 2 h at room temperature, and then washed 3×10 min. Reaction bands were visualized using a luminol-enhanced chemiluminescence (ECL) HRP substrate (Thermo Fisher Scientific). Each blot was then incubated with stripping buffer [2% SDS, 50 mM Tris-HCl (pH 6.8) and 100 mM β-mercaptoethanol] for 45 min at room temperature, and reprobed for other proteins, including actin or GADPH used as internal controls. Reaction product levels were quantified by scanning densitometry using ImageJ software (https://imagej.nih.gov/ij/) and normalized to the levels of actin or GAPDH (Lin et al., 2014a (link),b (link)).

Western blotting was performed as described previously (Lin et al., 2014b (link)). Protein content was determined using the BCA Protein Assay kit (Thermo Fisher Scientific). Equal amounts of total protein (30 µg tissue homogenate per lane) were subjected to 4-12% NuPAGE Gel for electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 3% nonfat milk and incubated with primary antibody overnight at 4°C. Blots were then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology) for 2 h at room temperature and then washed; reaction bands were visualized using a luminol-enhanced chemiluminescence (ECL) HRP substrate (Thermo Fisher Scientific). Each blot was then incubated with stripping buffer (2% SDS, 50 mM Tris, pH 6.8, and 100 mM β-mercaptoethanol) for 45 min at room temperature to remove the signals and reprobed for other proteins, including actin or GAPDH as an internal control. Reaction product levels were quantified by scanning densitometry using NIH ImageJ software (https://imagej.nih.gov/ij/) and normalized to the levels of actin and GAPDH.