Chemostar imager

Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma, Taufkirchen, Germany). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma, #T6199), IRX1 (Biozol, Eching, Germany, #DF3225), and IRX3 (Biozol, #MBS8223417). For loading control, blots were reversibly stained with Poinceau (Sigma) and the detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).
The enzyme-linked immunosorbent assay (ELISA) was used to quantify BMP2 protein levels in the supernatant of cell cultures. In addition, 2 × 10⁶ cells were cultured in 2 mL of fresh medium in a 24-well plate. After 24 h, 1 of mL medium was harvested and frozen in aliquots. Quantification was performed using the Quantikine ELISA BMP-2 kit (R & D Systems, #DBP200), as described by the company. Two biological replicates were analyzed in triplicate.

Western blots were generated by the semi-dry method, and protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma), NFKB1 (R&D Systems, Wiesbaden, Germany), and NFKB2 (Abcam, Cambridge, UK). The used antibodies were diluted as follows: alpha-Tubulin (TUBA) 1:1000, NFKB1 1:1000, NFKB2 1:500. For loading controls, blots were reversibly stained with Poinceau (Sigma), and detection of TUBA was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Western blot analyses were performed twice. Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).

Preparation of cell lysates, extraction of proteins, and immunoblotting were carried out as described earlier (32 (link), 34 (link)). Proteins were separated on TRIS-glycine gels (7.5% for NOS2 and 12% for Arginase 1) and transferred to PVDF membranes. Staining with appropriate primary and secondary antibodies was followed by signal visualization using the Chemo Star Imager (Intas Science Imaging Instruments, Göttingen, Germany). Densitometry of signals was done using ImageJ (Version 1.52a; Rasband, W., ImageJ, National Institutes of Health, USA, https://imagej.nih.gov/ij/).