Western Blot Analysis of Cellular Signaling

Lysates for western analyses were obtained by standard RIPA buffer-mediated lysis with the use of cell scrapers to facilitate the complete removal of the cells from the culture plate. RIPA buffer contained protease inhibitors (cOmplete Protease Inhibitor Cocktail; Roche #11 836 153 001; Sigma, Sofia, Bulgaria) and phosphatase inhibitors (PhosSTOP; Roche #04 906 837 001; Sigma, Sofia, Bulgaria). Antibodies for western analyses included: RON (C-20 clone; Cat#SC322, SCBT), HGFL (Cat#SC6088, SCBT), and C4-ACTIN (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA). Secondary antibodies were developed as described [6 (link)]. CM from R7 cells or BMDMs was assessed as per the manufacturer’s instructions using the Mouse Cytokine Array XL kit (R&D Systems, Minneapolis, MN, USA, Cat#ARY028). Densitometric analyses of the resulting signals were performed using VisionWorks 7 software (AnalitykJena, Thuringia, Germany).