Nupage tris hcl precast gels

Total protein from around 50 mg of flash-frozen mammary tumors was extracted with T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, St. Louis, MO, USA), according to the manufacturer’s protocol. Protein concentrations were ascertained utilizing a Bradford Assay, and denatured samples were subjected to electrophoresis on 4–15% NuPAGE Tris-HCl precast gels (Invitrogen, Waltham, MA, USA). Proteins were transferred onto nitrocellulose membranes and subsequently probed with antibodies to p21, p53, and BRCA2. Actβ was used as the loading control for each membrane. Antibody details can be found in Supplementary Table S2. Protein bands were visualized using Clarity Max™ Western ECL Blotting Substrates (Bio-Rad, Hercules, CA, USA) on a ChemiDoc™ XRS + System (Bio-Rad). Protein expression was quantified using ImageJ. For Western Blot experiments n = 6.

About 50 mg of frozen TNBC PDX tumors were used to extract total protein with T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s guidelines. The protein concentration was evaluated by Bradford Assay. The equal amount of denatured protein extracts was separated through electrophoresis in 4–15% NuPAGE Tris-HCl precast gels (Invitrogen, Waltham, MA, USA) and transferred onto nitrocellulose membranes. Membranes were then probed with primary antibodies including Cd74, Sat1, TAp63, Bcl-xL, Dnmt1, Dnmt3a, Dnmt3b, Tet2, Tet3, Hdac1, Hdac2, Hdac3, Hdac8 (Cell Signaling Technology, Danvers, MA, USA), Tet1, Ifi44, Fzd9, Wwc1 (Thermo Fisher Scientific, Waltham, MA, USA) and NF-κB and Lpl (Santa Cruz, Dallas, TX, USA). β-actin served as an internal control for each membrane. Immunoreactive bands were visualized using Clarity Max^TM Western ECL Blotting Substrates (Bio-Rad, Hercules, CA, USA) and images were captured using ChemiDoc^TM Imaging Systems (Bio-Rad, Hercules, CA, USA). The protein expression levels were quantified using Image J software (v1.53e).