Immobilon p transfer membranes

NTM cells were cultured in 60-mm dishes until they were confluent. The cells were serum-starved for 24 hours and treated with 100 ng/ml Wnt3a and/or 1 μg/ml sFRP1 for an additional 4, 24, or 48 hours. After treatment, the membrane protein fraction was isolated using the Eukaryotic Membrane Extraction Kit (Thermo Fisher Scientific), while total protein was isolated using the M-PER Mammalian Extraction Reagent (Thermo Fisher Scientific). Equal amounts of protein from each sample were separated using SDS-PAGE, transferred onto an Immobilon-P transfer membranes, blocked with 5% dry milk, and immunoblotted overnight at 4°C with rabbit anti-K-cadherin antibody (1:500; Abcam), rabbit anti-OB-cadherin antibody (1:500; Abcam), rabbit anti-β-catenin antibody (1:500; Cell Signaling), rabbit anti-GAPDH antibody (1:10,000; Cell Signaling), or mouse anti-Lamin A/C antibody (1:1000; Cell Signaling). The blots were then washed and incubated in secondary horse radish peroxidase (HRP)-linked anti-rabbit or anti-mouse IgG antibodies (1:5000; Cell Signaling Technology). Signals were developed using the Clarity Western ECL Blotting substrate (Bio-Rad). Images were taken using the Bio-Rad ChemiDoc imaging system (Bio-Rad). For some blots, densitometry was performed using ImageJ (National Institutes of Health, Bethesda, MA, USA).

Tibias were homogenized in RIPA lysis buffer including protease inhibitor (Roche, Mannheim, Germany) and centrifuged at 10,000× g for 10 min at 4 °C. The total protein levels were determined using a Bio-Rad Protein Assay Kit. The proteins were subjected to SDS-PAGE and transferred onto Immobilon-P transfer membranes, which were blocked with bovine serum albumin (5%) prior to incubation with specific primary antibodies raised against BMP-2, Wnt3a, RUNX2, or COL-1 (Abcam); p-SMAD 1/5/8 (Santa Cruz, Texas, USA); or p-ERK, p-p38, p-JNK, or β-actin (Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated with the appropriate secondary antibodies, either goat anti-rabbit IgG H&L (HRP) (Abcam) or goat anti-mouse IgG H&L (HRP) (Abcam). The antigen-antibody complexes were visualized using enhanced chemiluminescence. Densitometric analysis was performed using a C-DiGit Blot Scanner (Li-COR Biosciences, Lincoln, NE, USA).