Roti quant

Protein concentration was measured using the Bradford method with Rotiquant according to the supplier manual (Carl Roth, Karlsruhe, Germany). The assay was performed in a 96-well microplate format with a double determination at 450 and 590 nm [21 (link)]. Bovine serum albumin was used for calibration.

Protein concentration was measured by Bradford method using Roti‐Quant (Carl Roth GmbH) with bovine serum albumin as a standard. SDS–PAGE was performed according to standard procedures. Protein extracts were examined on 12 and 15% acrylamide gels. Western blot was performed using PVDF membranes (Millipore, Billerica, MA, USA), and specific antisera were used for protein immunodetection. HA‐tagged and TAP‐tagged proteins were detected by the use of monoclonal anti‐HA and PAP soluble complex antibodies (Sigma‐Aldrich), respectively. Enhanced chemiluminescence signals were detected by X‐ray films (Foma Bohemia, Hradec Kralove, Czech Republic), digitalized by Perfection V850 Pro scanner (EPSON, Long Beach, CA, USA) and quantified using ImageQuant TL (GE Healthcare) software. The images were processed using Photoshop CS4 (Adobe Systems, San Jose, CA, USA). The nomenclature of proteins is according to the Saccharomyces Genome Database (SGD). For ribosomal proteins, unified nomenclature was used according to 83.

To quantify the calcium deposition (mineralization) of osteogenic samples, murine MSCs and MC3T3 cells were harvested on day 14. After washing the samples twice with PBS and disrupting the monolayer with a cell scraper, calcium ions were dissolved from the extracellular matrix by shaking in 500 μl/well 0.5 N HCl at 4°C for 4 hr. Calcium content was determined in technical triplicates with the QuantiChrom calcium assay kit (BioAssay Systems, Hayward, CA) according to the manufacturer’s protocol. Protein content was measured in technical triplicates using Roti-Quant (Carl Roth, Karlsruhe, Germany) according to the manufacturer’s instructions. Calcium concentrations were normalized to the protein content.

Protein was isolated with 100 µL of CelLytic (C2978, Sigma Aldrich, St. Louis, MO, USA) supplemented with proteinase inhibitor (87786, Thermo Fisher Scientific, Waltham, MA, USA) and concentration was determined with RotiQuant (K015.3, Carl Roth, Karlsruhe, Germany) according to manufacture’s instructions. Equal amounts of protein were separated on 10% polyacrylamid gels. The proteins were transferred to a PVDF membrane with a pore size of 0.45 µm (T830.1, Carl Roth, Karlsruhe, Germany) for 90 min at 90 V. To prevent subsequent nonspecific binding of antibodies to the membrane, the blotted membrane was incubated for 1 h at room temperature in 5% milk powder (T145.3, Carl Roth, Karlsruhe, Germany) dissolved in Tris-buffered saline with Tween20 (TBS-T). ACTIN (E1C602-2, EnoGene, New York, NY, USA) and PTGS2 (PA5-16817, Thermo Fisher Scientific, Waltham, MA, USA) were used as primary antibodies. These were each incubated overnight at 4 °C and after washing three times in TBS-T, the membrane was incubated in the secondary antibody (611-1302, Rockland Immunochemicals, Gilbertsville, PA, USA) for 1 h at room temperature. After washing in TBS-T, Luminata Crescendo Western HRP Substrate (WBLUR0100, Sigma Aldrich, St. Louis, MO, USA) was added and the signal was digitized using the VWR Genoplex documentation system (VWR international, Radnor, PA, USA).

Cells were treated for 24 h with 10 µM NCT-503 or inactive NCT-503 control, and then lysed in CelLytic M provided in the citrate synthase assay kit (CS0720, Sigma-Aldrich, St. Louis, MO, USA). Aliquots of whole-cell extract containing equal total protein amounts were used for the citrate synthase activity assay according to the manufacturer’s instructions. Total protein was assessed by colorimetric protein quantification based on the Bradford assay, ROTI®Quant (Carl Roth, Karlsruhe, Germany).

THP‐1 macrophages (0.8 × 10⁶/well) were seeded in 6‐well plates and first treated with the indicated concentrations of piperine and pioglitazone for 24 h in FBS‐free RPMI‐1640 medium supplemented with 0.1% BSA and 10 μg/mL unesterified cholesterol. After incubation, cells were lysed with NP40 buffer (150 mM NaCl; 50 mM HEPES (pH 7.4); 1% NP40; 1% protease inhibitor Complete (Roche); 1% PMSF; 0.5% Na₃VO₄; 0.5% NaF) for 30 min at 4°C. The lysed cells were collected and centrifuged at 16 060 g for 20 min to collect the supernatant. Protein concentration was assessed with Bradford assay using Roti^®‐Quant from Carl Roth (#K015.1, Karlsruhe, Germany). Total protein (20 μg per sample) was loaded and separated via SDS‐PAGE. Expression levels of specific proteins were analyzed using antibodies against the indicated proteins and visualized with ECL reagent and a LAS‐3000 luminescent image analyzer (Fujifilm) with AIDA image analyzer 4.06 software (Raytest). A representative whole‐blot picture resulting from the detection of ABCA1 in the presence and absence of piperine is presented in the Supporting Information Fig. 1.