Journal: Experimental and Therapeutic Medicine

Article Title: IL-1β promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells via the BMP/Smad pathway within a certain concentration range

doi: 10.3892/etm.2020.9065

Figure Lengend Snippet: Effect of IL-1β on the BMP/Smad signaling pathway in mouse bone marrow mesenchymal stem cells. (A) Levels of p-Smad1/5, overall Smad1, Smad5 and Smad4 at 7 days were examined via Western blot analysis. (B) Quantitative assay of p-Smad1/5/GAPDH. * P<0.05, ** P<0.01, *** P<0.001. p-, phosphorylated; IL, interleukin; BMP, bone morphogenetic protein.

Article Snippet: After incubation in 5% skimmed milk for 1 h at 25˚C to block non-specific binding, the membranes were incubated with primary antibodies (1:1,000 dilution) against Smad1, Smad5, Smad4 and phosphorylated (p)-Smad1/5 (cat. no. 12656; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 5174; Cell Signaling Technology, Inc.) for 8 h at 4˚C.

Techniques: Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: IL-1β promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells via the BMP/Smad pathway within a certain concentration range

doi: 10.3892/etm.2020.9065

Figure Lengend Snippet: Effect of TGF-β/Smad inhibitor on the osteogenic differentiation of MBMMSCs. MBMMSCs were treated with osteogenic differentiation medium in the presence of 0.1 ng/ml IL-1β along with the inhibitor of TGF-β/Smad LDN193189. (A) Western blot analysis results for the levels of p-Smad1/5, and total Smad1, Smad5 and Smad4 at 7 days. (B) Quantitative results of p-Smad1/5/GAPDH. (C) Entire plate views of ALP staining at 7 days and alizarin red staining at 21 days. (D) Quantitative evaluation of ALP activity and alizarin red staining results. (E) mRNAs expression levels of RUNX2, ALP, COL1A1, BSP, BMP2, OCN and OPN at 7 days. Expression levels were normalized to GAPDH. *** P<0.001. p-, phosphorylated; IL, interleukin; ALP, alkaline phosphatase; BMP, bone morphogenetic protein; OCN, osteocalcin; OPN, osteopontin; BSP, bone sialoprotein; RUNX2, runt-related transcription factor 2; COL1A1, type I collagen; MBMMSCs, mouse bone marrow mesenchymal stem cells.

Article Snippet: After incubation in 5% skimmed milk for 1 h at 25˚C to block non-specific binding, the membranes were incubated with primary antibodies (1:1,000 dilution) against Smad1, Smad5, Smad4 and phosphorylated (p)-Smad1/5 (cat. no. 12656; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 5174; Cell Signaling Technology, Inc.) for 8 h at 4˚C.

Techniques: Western Blot, Staining, Activity Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: TGF-Beta Induces Activin A Production in Dermal Fibroblasts Derived from Patients with Fibrodysplasia Ossificans Progressiva

doi: 10.3390/ijms24032299

Figure Lengend Snippet: Expression of pSMAD1/5/9 in fibroblasts of FOP patients and controls by western blotting analysis. ( A ) Cells were serum-starved overnight before 60 min of stimulation with Activin A and ( B ) serum-starved overnight before 90 min of stimulation with BMP4. Actin was used to determine equal protein loading. −FBS are serum-starved unstimulated fibroblasts.

Article Snippet: Primary antibodies against phospho-SMAD3 (cat#52903, Abcam), phospho-SMAD1/5/9 (AB3848-I, Sigma-Aldrich), and Actin (Cat#ab14128, Abcam) were used for overnight incubation.

Techniques: Expressing, Western Blot