Quantification of RANKL Protein Levels

This factor in MSCs (6 BCPs and 5 HVs) was analyzed by Western Blot. Briefly, cells were lysed in RIPA buffer [Tris (pH=7.5) 25 mM, NaCl 360 mM, nonidet P-40 2.5%, sodium deoxycholate 1%, SDS 0.25%, sodium vanadate 5 mM], supplemented with proteases and phosphatases inhibitor cocktail (P8340, Sigma) at 4°C for 30 min. After centrifugation for 30 min at 16,000 g at 4°C, pellets were frozen and conserved at -80°C until use. SDS-PAGE electrophoresis was performed using 20 µg of denatured protein, later transferred onto a nitrocellulose membrane (RPN3032D, Amersham). The membrane was stained with 0.2% Ponceau S in 0.5% acetic acid to verify the integrity of the transferred proteins. After blocking with 5% skim milk in T20-TBS, the membranes were incubated with primary Abs against human RANKL (1:200, mouse IgG2b, MAB626, Clone: 70525, R&D Systems) and β-Actin (1:2000, mouse IgG1, ab6276, Clone: AC-15, Abcam). Goat anti mouse Ig conjugated to horseradish peroxidase was used as secondary Ab (goat IgG, HAF007, R&D Systems). Hybridizing bands were visualized using ECL™ Prime Western Blotting System (GERPN2232, Sigma) and were digitalized using the image analysis system (G Box Chemi XT16, Syngene, USA). Relative RANKL quantification was performed by normalization with β-Actin signal and expressed as mean relative density using ImageJ analysis software (NIH).