Of protease and phosphatase inhibitors

Immediately after collection, the hypothalamus or BAT was homogenized in RIPA buffer (Sigma) containing a cocktail of protease and phosphatase inhibitors (1:100, Sigma), centrifuged (14,000 RPM, 4°C for 20 min) and the supernatants were retained. After determination of total protein concentration (Pierce BCA Protein Assay, Thermo Scientific) 50 µg of total protein was loaded in a 10% SDS-PAGE gel and finally transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% bovine serum albumin and incubated overnight at 4°C using commercially available primary antibodies (1:1,000) to identify pSTAT3^Tyr705 (Cell Signaling), UCP1 (Santa Cruz), or β-Actin (Sigma). Next, we incubated the membranes for 60 min in 1:20,000 secondary antibody (IRDye 800CW, Li-COR). Proteins were detected by fluorescence, analyzed using the Li-COR Odyssey system (Li-COR), and normalized to β-Actin or Ponceau staining (30 (link)).

In the first stage, samples of yellow ligamentum flavum collected from patients of the study group and participants of the control group were rinsed with a PBS solution and then placed in a new Eppendorf tube to which 0.50 ml of radioimmunoprecipitation assay buffer (RIPA; Sigma Aldrich St. Louis, MO, USA) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma Aldrich St. Louis, MO, USA). The samples were then homogenized using a hand-held homogenizer (T18 Digital Ultra-Turrax, IKA Polska Sp. z o. o., Warsaw, Poland) until no solid fragments were visible. After this step, the tubes were placed on ice and gently mixed on a rocker for 60 minutes, after which the samples were centrifuged and the supernatant was collected and stored at −80°C until further analysis.
After thawing, the total protein concentration in the preparations was determined using the KIT bicinchoninic acid assay (BCA; Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s guidelines. Protein concentrations ranged from 20 to 100 μg of total protein. All protein concentration measurements were calculated using a standard curve based on standard solutions of bovine serum albumin (BSA; including a set of six standard points − 0, 250, 500, 1000, 1500, and 2000 µg/ml) (Sigma Aldrich St. Louis, MO, USA).

Proteins were extracted with RIPA buffer (Sigma-Aldrich) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Briefly, cells were trypsinized from culture dishes, centrifuged, and washed once with cold PBS. Cell pellets were then resuspended and incubated in RIPA buffer for 20 min on ice and sonicated with 3 pulses 10 sec each at 10% amplitude (Branson Digital Sonifier, Danbury, CT, USA). After centrifugation, supernatants were denatured in Laemmli loading buffer for 10 minutes at 98°C. Total protein (20 μg) was separated by SDS-PAGE and transferred to PVDF membranes which were subsequently probed with anti-Nanog (Bethyl, Montgomery TX, USA), anti-Oct4 (BD Transduction, San José, CA, USA), anti-Pdx1 (Abcam, Cambridge, UK), anti-Sox17 (Millipore, Billerica, MA, USA), or anti-Gata4 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-β-actin (Sigma-Aldrich) as loading control.