Journal: Molecular Medicine Reports

Article Title: Bone morphogenetic protein-9 promotes the differentiation of mouse spleen macrophages into osteoclasts via the ALK1 receptor and ERK 1/2 pathways in vitro

doi: 10.3892/mmr.2016.5803

Figure Lengend Snippet: Effects of BMP-9 on the expression of BMP receptors. (A) Expression of BMPR-IA, BMPR-IB, BMPR-II and ALK1 receptors in cells treated with BMP-9 (100 ng/ml) using ELISA. **P<0.01 and *P<0.05, vs. other receptors. (B) Immunofluorescence of (a) ALKI receptor, (b) counterstaining with DAPI and (c) the two stains merged in cells without BMP-9 treatment. Immunofluorescence of (d) ALKI receptor (e) counterstaining with DAPI and (f) the two stains merged in cells treated with BMP-9 (100 ng/ml). Positive expression of ALK1 receptor is indicated by white arrows. BMP-9, bone morphogenetic protein-9; BMPR, BMP receptor; ALK1, anaplastic lymphoma kinase 1; OD, optical density.

Article Snippet: The cells were fixed with 4% paraformaldehyde and blocked with 5% BSA, and were then incubated overnight at 4°C with the following primary antibody: Polyclonal goat anti-mouse ALK1 immunoglobulin (Ig) G (1:100; cat. no. 770-MA; R&D Systems, Inc., Minneapolis, MN, USA), quenched with glycine (0.1 M) for 1 h, incubated with secondary antibodies (polyclonal donkey anti-goat IgG; 1:200; cat. no. NL003; R&D Systems, Inc.) for 2 h at room temperature, and then washed three times with PBS.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Molecular Medicine Reports

Article Title: Bone morphogenetic protein-9 promotes the differentiation of mouse spleen macrophages into osteoclasts via the ALK1 receptor and ERK 1/2 pathways in vitro

doi: 10.3892/mmr.2016.5803

Figure Lengend Snippet: Effects of BMP-9 and ALK1 receptor on the cell signal transduction pathway. (A) Western blot of the phosphorylation of ERK1/2 in cells treated with BMP-9 (100 ng/ml) and (B) quantification (*P<0.01, vs. 0 min). (C) Western blot of the phosphorylation of Smad2 in cells treated with BMP-9 (100 ng/ml) and (D) quantification (P>0.01, vs. 0 min). (E) Western blot of the phosphorylation of ERK1/2 in cells pre-treated with siRNA-ALK1 and (F) quantification (*P<0.01, vs. BMP-9). ERK1/2, extracellular signal-regulated kinase 1/2; p-ERK1/2, phosphorylated ERK1/2; Smad 2, small mothers against decapentaplegic 2; p-Smad2, phosphorylated Smad 2; BMP-9, bone morphogenetic protein-9; ALK1, anaplastic lymphoma kinase 1; siRNA, small interfering RNA.

Article Snippet: The cells were fixed with 4% paraformaldehyde and blocked with 5% BSA, and were then incubated overnight at 4°C with the following primary antibody: Polyclonal goat anti-mouse ALK1 immunoglobulin (Ig) G (1:100; cat. no. 770-MA; R&D Systems, Inc., Minneapolis, MN, USA), quenched with glycine (0.1 M) for 1 h, incubated with secondary antibodies (polyclonal donkey anti-goat IgG; 1:200; cat. no. NL003; R&D Systems, Inc.) for 2 h at room temperature, and then washed three times with PBS.

Techniques: Transduction, Western Blot, Phospho-proteomics, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Bone morphogenetic protein-9 promotes the differentiation of mouse spleen macrophages into osteoclasts via the ALK1 receptor and ERK 1/2 pathways in vitro

doi: 10.3892/mmr.2016.5803

Figure Lengend Snippet: Effects of the ALK1 receptor and ERK1/2 pathways on BMP-9-induced osteoclast differentiation (A) TRAP staining of cells (a) induced by BMP-9 (100 ng/ml) and RANKL (100 ng/ml), and (b) pre-transfected with siRNA-ALK1 or (c) cultured with U0126 (1,000 nmol/l) prior to BMP-9+RANKL. (Phase contrast microscope; magnification, ×20). White arrows indicate TRAP-positive cells. (B) Protein expression of CTR, determined using an enzyme-linked immunosorbent assay, in cells treated with BMP-9 (100 ng/ml) and RANKL (100 ng/ml), transfectecd with siRNA-ALK1 or treated with U0126 (1,000 nmol/l). *P<0.01, vs. BMP-9+RANKL group. BMP-9, bone morphogenetic protein-9; ALK1, anaplastic lymphoma kinase 1; RANKL, receptor activator for nuclear factor-κb ligand; CTR, calcitonin receptor; siRNA, small interfering RNA; OD, optical density; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: The cells were fixed with 4% paraformaldehyde and blocked with 5% BSA, and were then incubated overnight at 4°C with the following primary antibody: Polyclonal goat anti-mouse ALK1 immunoglobulin (Ig) G (1:100; cat. no. 770-MA; R&D Systems, Inc., Minneapolis, MN, USA), quenched with glycine (0.1 M) for 1 h, incubated with secondary antibodies (polyclonal donkey anti-goat IgG; 1:200; cat. no. NL003; R&D Systems, Inc.) for 2 h at room temperature, and then washed three times with PBS.

Techniques: Staining, Transfection, Cell Culture, Microscopy, Expressing, Enzyme-linked Immunosorbent Assay, Small Interfering RNA