Bca protein assay

The activity of alkaline phosphatase (ALP), a widely accepted early marker of osteogenesis in bone-forming cells,^{28 (link)} was assessed on cells grown for 14 days on glass or P3HT substrates, under either standard culture conditions or osteogenic stimuli (10 nM dexamethasone, 10 mM glycerol-2-phosphate, 150 mM l-ascorbic acid and 10 nM cholecalciferol, Sigma Aldrich). Briefly, cells were washed in phosphate buffer saline and lysed in 50 μL 0.1% Triton X-100 (Sigma Aldrich). ALP activity was assessed through a colorimetric assay based on the conversion of p-nitrophenyl phosphate into p-nitrophenol, as exhaustively described by Canciani et al.^{29 (link)} The enzymatic activity (U) was inferred based on the amount of produced p-nitrophenol and the reaction time, then normalized with respect to each sample's protein concentration determined by BCA™ Protein Assay (Merck) and expressed as U μg⁻¹.

The T-PER^TM Tissue Protein Extraction Reagent (Thermo Fisher Scientific) combined with the Halt^TM protease inhibitor cocktail (Thermo Fisher Scientific) was used to lyse the HCT116 cells. The lysate was centrifuged at 17,000 × g for 20 min at 4°C, and the supernatant was recovered as a detergent soluble fraction as previously described.⁶⁶ (link) The insoluble pellet was dissolved in T-PER^TM Tissue Protein Extraction Reagent supplemented with 2% sodium dodecyl sulfate and centrifuged at 17,000 × g for 20 min at 4°C. The supernatant was separated into insoluble fractions. The concentration of each protein fraction was determined using the BCA protein assay (Merck, Shanghai, China) and analyzed using western blotting.

Total protein was isolated from BEAS-2B cells using RIPA Lysis Buffer (cat. no. 20-188; Merck KGaA), then quantified with BCA Protein Assay (cat. no. P0012; Beyotime Institute of Biotechnology) and the protein was denatured in a 95˚C water bath for 5 min. Proteins (30 µg/lane) were separated by 10% SDS-PAGE, transferred onto PVDF membranes (Millipore; Merck KGaA), blocked with 5% non-fat milk for 2 h at room temperature and incubated overnight at 4˚C with the following primary antibodies: Anti-ETS1 (cat. no. ab220361; Abcam; 1/1,000); anti-phosphorylated (p)-ERK1/2 (cat. no ab201015; Abcam; 1/1,000); anti-ERK1/2 (cat. no. ab184699; Abcam; 1/8,000) and anti-GAPDH (cat. no. ab181602; Abcam; 1/10,000). This was followed by incubation with rabbit anti-mouse horseradish peroxidase-conjugated secondary antibodies (cat. no. ab6721; Abcam; 1/5,000) for 60 min at room temperature. Protein bands were scanned and imaged using Immobilon^® Western HRP Substrate ECL chemiluminescence reagent (cat. no. WBKLS0500; Merck KGaA). Densitometry analysis was performed using ImageJ software (Version 1.53e; National Institutes of Health) with GAPDH as the loading control.