Quick start bradford dye reagent

Malonyl coenzyme A (malonyl-CoA), acyl-CoAs, fatty acids, NADH, NADPH, and antibiotics were purchased from Sigma-Aldrich (St. Louis, MO, USA); Takara Biotechnology Co., Ltd. (Dalian, China) provided molecular biology reagents; the Ni²⁺-agarose columns were purchased from Invitrogen (Shanghai, China); American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA) provided sodium [1-¹⁴C] acetate (specific activity, 50 mCi/mM); and Bio-Rad (Hercules, CA, USA) provided the Quick Start Bradford dye reagent. All other reagents were of the highest available quality.

Urine protein levels were measured using the Quick Start Bradford Dye Reagent (BioRAD). Serum creatinine was measured using the Enzymatic Creatinine Liquid Color Reagent (Stanbio Laboratory, Boerne, TX), according to the manufacturer's instructions.

Proteins were extracted from two independent batches of scCM (~ 100 µg each) in a 1.5 M Tris–HCl buffer (pH 8.5) containing 7 M guanidine hydrochloride (GuHCl), 20 mM ethylenediaminetetraacetate (EDTA), and 0.5 M dithiothreitol (DTT), and incubated for 1 h at 60 °C. The sulfhydryl groups of the proteins were carbamidomethylated with iodoacetamide used in a 2.5-fold excess (w/w) to DTT in the dark (20 min, RT). The suspension was centrifuged at 11.000 g for 15 min at 4 °C, and the supernatant was collected. Protein concentration was measured using the Quick start Bradford dye reagent (Biorad, Hercules, USA) with BSA as a standard protein. For each sample, 100 μg of protein was precipitated in 80% acetone overnight at − 20 °C. After 15 min of centrifugation at 11.000 g, the pellet was enzymatically digested overnight using a sequencing grade modified trypsin (Promega, Madison, USA) with an enzyme/substrate ratio of 1/50 at 37 °C in 25 mM ammonium bicarbonate (NH₄HCO₃). The reaction was stopped by adding formic acid to a final concentration of 0.1% (v/v). Peptides were extracted using the HyperSep SpinTip Microscale C18 (Thermo Fisher Scientific, USA), and the peptides’ concentration was measured using the Quantitative Colorimetric Peptide Assay (Thermo Fisher Scientific, USA).