Supersignal

Cells of the MG-63 and MDA-MB-231 cell line were treated with 0 (control cells; C) or 100 mN leptin (leptin group). Cell lysis buffer (cat. no. P0013K; Beyotime Institute of Biotechnology, Haimen, China) was then used to extract total protein from these cells. Total protein concentration was determined via bicinchoninic acid assay. A total of 20 µg protein from each sample was subjected to electrophoresis using 10% SDS-PAGE, and were subsequently transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). Blocking was performed by incubating membranes with 5% skimmed milk at room temperature for 2 h. Following washing, membranes were incubated with primary antibodies against β-catenin (1:1,200; cat. no. ab16051; Abcam, Cambridge, UK) and endogenous control β-actin (1:1,000; cat. no. SAB5500001; Sigma-Aldrich; Merck KGaA) overnight at 4°C. Following washing, membranes were incubated with horseradish peroxidase conjugated anti-rabbit immunoglobulin G secondary antibodies (1:1,000; cat. no. MBS435036; MyBioSource, San Diego, CA, USA) at 37°C for 1 h. Enhanced chemiluminescence (SuperSignal; Thermo Fisher Scientific, Inc.) was performed to detect the signals and Quantity One^® 1-D Analysis Software V. 4.6.7 (Bio-Rad Laboratories, Inc.) was used to measure grayscale. This experiment was repeated three times.

Protein lysate was mixed with NuPAGE loading buffer and NuPAGE reducing agent (Invitrogen) and boiled for 10 min at 95°C. 5–15 µg of protein (lysed as described in the CoIP section) or total CoIP elution was loaded in 4–12% Bis-Tris gels (NuPAGE Novex; Invitrogen). After, electrophoresis gels were transferred to nitrocellulose membranes (GE Healthcare) and blocked with 5% milk in 0.1% PBS with Tween 20 (PBST). Membranes were incubated overnight at 4°C with the previously mentioned antibodies diluted in 5% nonfat dry milk in PBST. Upon three PBST washes, membranes were incubated with peroxidase-conjugated secondary antibodies (Dako) for 2 h at 20–26°C and developed with ECL (GE Healthcare) or Supersignal (Thermo Fisher Scientific) depending on the antibody. Western blot analysis was performed measuring the integrated density of the corresponding bands and normalizing them to β-actin (Sigma-Aldrich) control or CoIP control antibody, which is an antibody against the protein for which the pull-down has been done (RXR-γ in most cases).

Following PK digestion, the samples were mixed with an equal volume of a 2-butanol:methanol mixture (5:1), and PrP^Sc was precipitated by 20,000 ×g centrifugation. The pellets were resuspended in Laemmli sample buffer and boiled for 5 min. Samples were electrophoresed on NuPAGE Novex 12% Bis-Tris gels and NuPAGE MOPS-SDS running buffer in accordance with the manufacturer's instructions (Life Techonologies, Carlsbad, CA, USA). The proteins were transferred onto an Immobilon-P membrane (Millipore, Billerica, MA, USA). The blotted membrane was incubated with anti-PrP monoclonal antibodies T2, 6H4 (Prionics, Zurich, Switzerland), and SAF84 (Bertin Pharma, Montigny le Bretonneux, France) at 4°C overnight. After washing with PBS containing Tween 20 twice, the membrane was incubated with HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 60 min at R/T. Signals were developed with a chemiluminescent substrate (SuperSignal; Thermo Fisher Scientific). For semi-quantitation, blots were imaged using a Fluorchem system (Alpha Innotech, San Leandro, CA, USA) and analyzed using image reader software (AlphaEaseFC; Alpha Innotech) according to the manufacturer's instructions.