Bca protein assay

Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Complete™, Mini, EDTA-free, Sigma-Aldrich). Total protein content was determined by BCA Protein Assay (Thermo Scientific). Total protein content was determined by BCA Protein Assay (Thermo Scientific). Samples were loaded on 8%–12% NuPAGE gel (Invitrogen, Carlsbad, CA, USA). Electrophoresis was performed at 200 V for 1 h. Transfer of proteins to PolyScreen PV transfer membrane (PerkinElmer Life Sciences, Boston, MA, USA) was performed for 2 h, at 25 volts. Membrane was blocked overnight in 5% non-fat dry milk in phosphate buffered saline. Protein levels of heme oxygenase 1 (HO-1) and NQO1 were detected using rabbit monoclonal anti-mouse antibodies, following manufacturer recommended dilutions (Abcam, Cambridge, MA, USA). Peroxidase-conjugated Donkey anti-rabbit IgG was used as a secondary antibody. (Jackson Laboratories, West Grove, PA, USA). Membranes were developed using Western Lighting Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA, USA) and bands were visualized on standard X-ray film (HyBlot CL, Denville Scientific Inc., Metuchen, NJ, USA). Densitometric analysis of the bands was performed using ImageJ (NIH) software.

To evaluate the expression of MMPs and TIMPs, LV samples from the peri infarct zone where the CSps had been injected were homogenized and processed as previously described with small modifications [12] (link), [13] (link). More specifically, LV tissues (n = 3 in each group) were homogenized in tissue lysis buffer (Thermo Scientific) without protease inhibitors and stored at −80°C. Final protein concentration was quantified with a standardized colorimetric assay (BCA Protein Assay, Thermo Scientific, Rockford, Illinois). Gelatin zymography was used to determine MMP2 and MMP9 pro form and active form while the rest of the MMPs and TIMPs of this study were evaluated by immunoblotting. Both zymograms and immunoblots were analyzed by densitometry Protein level in the dermal fibroblasts used in the in vitro cocultures was also determined by BCA Protein Assay (Thermo Scientific). Rb anti human col1 (Abcam) was used for the collagen detection.

Cells or tumors were lysed with Laemmli lysis buffer. Protein concentration was estimated using the BCA Protein Assay (Thermo Scientific). Equal amounts of protein were separated by SDS-PAGE and transferred onto nitrocellulose (Hybond, Amersham). The membrane was blocked with 5% skim milk in TBS-T for at least 60 min at RT. Primary antibodies were used as detailed in Supplementary Table S2. All secondary antibodies conjugated with horseradish peroxidase (Dianova) were used in dilutions of 1:5000–1:10000 in 5% skim milk (TBS-T). For the extraction of the nuclei, trypsinized cells were resuspended in cold DMEM + 10% FCS and centrifuged for 5 min at 300×g, 4°C. Cells pellets were washed with ice cold PBS, resuspended in Nuclei-Buffer I (10 mM Tris-HCl pH 7.9; 10 mM KCl; 15 mM MgCl₂) and incubated for 10 min on ice. After centifugation (1000×g/ 2 min/4°C), the nuclei were lysed in Laemmli lysis buffer supplemented with 1 μl Benzonase (Roche) and incubated for 5 min at 95°C. Protein concentration was estimated using the BCA Protein Assay (Thermo Scientific). All buffers used here were supplemented with protease (cOmplete mini EDTA free, Roche) and phosphatase inhibitors (2 mM Na₃VO₄ and 5 mM NaF). For protein quantification, signal acquisition and measurement was performed on a Gel Doc XR+ System (BioRad).