Horseradish peroxidase hrp conjugated anti goat or rabbit igg

Experimental cells were collected, homogenized, and lysed with lysis buffer (Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Samples of 25 μg protein were fractionated by SDS-PAGE in 4–20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% nonfat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4°C in blocking solution containing rabbit primary antibodies against KEAP1 (D6B12, Cell Signaling Technology, Boston, MA, USA). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase- (HRP-) conjugated anti-goat or rabbit IgG (1 : 2000; Santa Cruz Biotechnology, Inc.) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of western blots were captured by GE ImageQuant.

Cell extractions for protein assessment studies were performed as previously described [12 (link)]. Briefly, cells were lysed with Cell Lysis Buffer (#9803, Cell Signaling Technology, Boston, MA, USA) containing 0.5 mM of phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was standardized by BCA protein assay (Thermo Fisher Scientific, Grand Island, NY, USA). Samples (25 μg) were separated by SDS-PAGE in 4% – 20% gradient Tris-glycine precast gels (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated for 1 hour in blocking solution containing 5% non-fat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4 C in the blocking buffer containing rabbit primary antibodies against CD36 (Abcam, ab133625, 1:500). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG (1:2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of western blots were captured by GE imageQuant imager. Relative band intensities were analyzed using Image J software and normalized to GAPDH.