Trans blot turbo pvdf membrane

The protein expression of RUNX2, ALPL and BMPR1A was detected by western blotting on day 7 (2 days of co-culture). Cells were lysed with 150 μL of buffer constituted by 1 × protease inhibitor mixture (Roche Applied Science), 1 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich) and 25 mM MG132 proteasome inhibitor (Roche Applied Science). Briefly, 50 μg of total protein was denatured, separated in 10% SDS polyacrylamide electrophoresis gel and transferred to a trans-blot turbo PVDF membrane (Bio-Rad Laboratories). The membrane was blocked for 1 hour in Tris-buffered saline with 0.1% Tween® 20 (Sigma-Aldrich), containing 5% nonfat powdered milk (Bio-Rad Laboratories), probed with primary antibodies anti-RUNX2 (1:2000, Cell Signaling), anti-ALPL (1:3000, Abcam) and anti-BMPR1A (1:250, Sigma-Aldrich) overnight at 4°C, and incubated with secondary horseradish peroxidase-conjugated antibody goat anti-rabbit IgG (1:2000, Cell Signaling) for 1 hour at room temperature. The proteins were detected using Clarity Western ECL Substrate (Bio-Rad Laboratories) and the images were acquired using G-Box gel imaging (Syngene, Cambridge, UK). The proteins were quantified (n = 3) by counting pixels through Image-J software (National Institutes of Health, Bethesda, Maryland, USA), normalized to GAPDH (Santa Cruz Technology) and calibrated by non-co-cultured osteoblasts grown on Ti Control.

Cells were washed twice with ice-cold PBS and lysed with RIPA buffer (Life Technologies, Carlsbad, CA) supplemented with protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), 1mM NaVO4, and 1mM NaF as described previously [22 (link)]. Lysates were centrifuged at 10,000 rpm for 10 min at 4 °C, and supernatants were collected and protein quantification was performed using Bradford protein assay (Thermo Scientific, Tewksbury, MA) with BSA as the standard. Lysates were denatured using β-mercaptoethanol (Life Technologies) and Laemmli sample buffer (Bio-Rad, Waltham, MA) and heated at 70 °C for 10 min, electrophoresed in Mini Protean TGX 4–15% gels (Bio-Rad), transferred on Trans Blot Turbo PVDF membrane (Bio-Rad) and immunoblotted with specific primary antibodies. Primary antibody binding was detected using HRP-conjugated secondary antibodies (Santa Cruz, Dallas, TX) and ECL Prime Western Blot reagents (Amersham/GE Healthcare, Pittsburgh, PA) as chemiluminescence substrates. The immunoblots were analyzed and quantified using ImageJ software.