Pierce 660 nm assay kit

For this study, we performed the tandem mass tagging (TMT)-based analysis as we described in previous papers [15 (link)]. In brief, cellular proteins from 25μM pioglitazone-treated and control TRT-HU1 cells were extracted using a 4% sodium dodecyl sulfate-containing buffer. The protein concentration was measured using the Pierce 660nm Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The kit was carried out using 60 μg of protein from each sample. Samples were digested with trypsin via filter-aided sample preparation and labeled with TMT6plex reagents in parallel. Then, the peptides were reconstituted and desalted via C18 spin columns (Thermo Fisher Scientific). High-pH reversed-phase liquid chromatography using an Ultimate 3000 XRS System (Thermo Fisher Scientific) was used to separate samples. Peptides with >30% precursor ion interference were excluded to minimize inaccurate quantifications caused by precursor ion interference and Proteome Discoverer was used.

Lysis was performed in all cell lines with Passive Lysis Buffer (Promega) and then cells were subjected to a freeze–thaw cycle at −80°C to 37°C and centrifuged at maximum speed for 5 min. The cell lysates were used to determine luciferase and β-gal activities with the Dual-Luciferase Reporter Assay System (Promega) and Beta-Glo Assay System (Promega), respectively, using the Lucy 2 (Anthos Labtec) or GloMax 96 Microplate (Promega) luminometers, according to the manufacturer's standard protocol. When indicated, total protein content was determined with the Pierce 660 nm Assay Kit (Thermo Scientific) using NanoDrop 2000 (Thermo Scientific). The luciferase values are presented as the units of FLuc or RLuc after being normalized to β-gal or, alternatively, per µg of total protein. Each value was derived from at least three independent experiments.