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Design and validation of bone-targeted Bmp2. a. Schematic representation of the Bmp2/D-Bmp2 plasmid constructs and the structure predicted by AlphaFold3. b. Immunofluorescence staining of HEK-293T cells transfected with the plasmids: phalloidin (green), DAPI (blue), and Alexa Fluor 647-conjugated anti-Flag antibodies (red). Scale bar: 10 μm. c. SDS-PAGE of purified proteins. d. Western blot validation of protein expression. e. Bmp2 activity reporter assay: schematic of the luciferase reporter system (left), representative fluorescence images, and statistical analysis of firefly luciferase activity by in vivo imaging system <t>(IVIS)</t> (a.u.: arbitrary units) (right, n = 3 per group). f. qPCR analysis of Bmp2 signaling pathway-related mRNA levels in MC3T3-E1 cells treated with the Bmp2 or D-Bmp2 protein (n = 3 per group). g, h. Western blot (g) and quantification of Bmp2 pathway-related protein expression in treated MC3T3-E1 cells (h) (n = 3 per group). i. Schematic of the HA-coated ELISA plate and HA-binding affinity assay (n = 3 per group). j. Tissue-specific binding assay of Bmp2 or D-Bmp2: Mouse muscle slices (left, scale bar: 100 μm) and undecalcified femur slices (right, scale bar: 200 μm) stained with AF647-conjugated anti-Flag antibodies. The data are presented as the means ± standard deviations (SDs). One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.
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Design and validation of bone-targeted Bmp2. a. Schematic representation of the Bmp2/D-Bmp2 plasmid constructs and the structure predicted by AlphaFold3. b. Immunofluorescence staining of HEK-293T cells transfected with the plasmids: phalloidin (green), DAPI (blue), and Alexa Fluor 647-conjugated anti-Flag antibodies (red). Scale bar: 10 μm. c. SDS-PAGE of purified proteins. d. Western blot validation of protein expression. e. Bmp2 activity reporter assay: schematic of the luciferase reporter system (left), representative fluorescence images, and statistical analysis of firefly luciferase activity by in vivo imaging system <t>(IVIS)</t> (a.u.: arbitrary units) (right, n = 3 per group). f. qPCR analysis of Bmp2 signaling pathway-related mRNA levels in MC3T3-E1 cells treated with the Bmp2 or D-Bmp2 protein (n = 3 per group). g, h. Western blot (g) and quantification of Bmp2 pathway-related protein expression in treated MC3T3-E1 cells (h) (n = 3 per group). i. Schematic of the HA-coated ELISA plate and HA-binding affinity assay (n = 3 per group). j. Tissue-specific binding assay of Bmp2 or D-Bmp2: Mouse muscle slices (left, scale bar: 100 μm) and undecalcified femur slices (right, scale bar: 200 μm) stained with AF647-conjugated anti-Flag antibodies. The data are presented as the means ± standard deviations (SDs). One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.
Ivis Lumina Lt Series Iii Imaging System, supplied by Revvity, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Design and validation of bone-targeted Bmp2. a. Schematic representation of the Bmp2/D-Bmp2 plasmid constructs and the structure predicted by AlphaFold3. b. Immunofluorescence staining of HEK-293T cells transfected with the plasmids: phalloidin (green), DAPI (blue), and Alexa Fluor 647-conjugated anti-Flag antibodies (red). Scale bar: 10 μm. c. SDS-PAGE of purified proteins. d. Western blot validation of protein expression. e. Bmp2 activity reporter assay: schematic of the luciferase reporter system (left), representative fluorescence images, and statistical analysis of firefly luciferase activity by in vivo imaging system <t>(IVIS)</t> (a.u.: arbitrary units) (right, n = 3 per group). f. qPCR analysis of Bmp2 signaling pathway-related mRNA levels in MC3T3-E1 cells treated with the Bmp2 or D-Bmp2 protein (n = 3 per group). g, h. Western blot (g) and quantification of Bmp2 pathway-related protein expression in treated MC3T3-E1 cells (h) (n = 3 per group). i. Schematic of the HA-coated ELISA plate and HA-binding affinity assay (n = 3 per group). j. Tissue-specific binding assay of Bmp2 or D-Bmp2: Mouse muscle slices (left, scale bar: 100 μm) and undecalcified femur slices (right, scale bar: 200 μm) stained with AF647-conjugated anti-Flag antibodies. The data are presented as the means ± standard deviations (SDs). One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.
Ivis Spectrum, supplied by Revvity, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Design and validation of bone-targeted Bmp2. a. Schematic representation of the Bmp2/D-Bmp2 plasmid constructs and the structure predicted by AlphaFold3. b. Immunofluorescence staining of HEK-293T cells transfected with the plasmids: phalloidin (green), DAPI (blue), and Alexa Fluor 647-conjugated anti-Flag antibodies (red). Scale bar: 10 μm. c. SDS-PAGE of purified proteins. d. Western blot validation of protein expression. e. Bmp2 activity reporter assay: schematic of the luciferase reporter system (left), representative fluorescence images, and statistical analysis of firefly luciferase activity by in vivo imaging system <t>(IVIS)</t> (a.u.: arbitrary units) (right, n = 3 per group). f. qPCR analysis of Bmp2 signaling pathway-related mRNA levels in MC3T3-E1 cells treated with the Bmp2 or D-Bmp2 protein (n = 3 per group). g, h. Western blot (g) and quantification of Bmp2 pathway-related protein expression in treated MC3T3-E1 cells (h) (n = 3 per group). i. Schematic of the HA-coated ELISA plate and HA-binding affinity assay (n = 3 per group). j. Tissue-specific binding assay of Bmp2 or D-Bmp2: Mouse muscle slices (left, scale bar: 100 μm) and undecalcified femur slices (right, scale bar: 200 μm) stained with AF647-conjugated anti-Flag antibodies. The data are presented as the means ± standard deviations (SDs). One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.
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Design and validation of bone-targeted Bmp2. a. Schematic representation of the Bmp2/D-Bmp2 plasmid constructs and the structure predicted by AlphaFold3. b. Immunofluorescence staining of HEK-293T cells transfected with the plasmids: phalloidin (green), DAPI (blue), and Alexa Fluor 647-conjugated anti-Flag antibodies (red). Scale bar: 10 μm. c. SDS-PAGE of purified proteins. d. Western blot validation of protein expression. e. Bmp2 activity reporter assay: schematic of the luciferase reporter system (left), representative fluorescence images, and statistical analysis of firefly luciferase activity by in vivo imaging system <t>(IVIS)</t> (a.u.: arbitrary units) (right, n = 3 per group). f. qPCR analysis of Bmp2 signaling pathway-related mRNA levels in MC3T3-E1 cells treated with the Bmp2 or D-Bmp2 protein (n = 3 per group). g, h. Western blot (g) and quantification of Bmp2 pathway-related protein expression in treated MC3T3-E1 cells (h) (n = 3 per group). i. Schematic of the HA-coated ELISA plate and HA-binding affinity assay (n = 3 per group). j. Tissue-specific binding assay of Bmp2 or D-Bmp2: Mouse muscle slices (left, scale bar: 100 μm) and undecalcified femur slices (right, scale bar: 200 μm) stained with AF647-conjugated anti-Flag antibodies. The data are presented as the means ± standard deviations (SDs). One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.
Ivis Lumina K Series Iii Imaging System, supplied by Revvity, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ivis lumina ii in vivo imaging system  (Revvity)
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Design and validation of bone-targeted Bmp2. a. Schematic representation of the Bmp2/D-Bmp2 plasmid constructs and the structure predicted by AlphaFold3. b. Immunofluorescence staining of HEK-293T cells transfected with the plasmids: phalloidin (green), DAPI (blue), and Alexa Fluor 647-conjugated anti-Flag antibodies (red). Scale bar: 10 μm. c. SDS-PAGE of purified proteins. d. Western blot validation of protein expression. e. Bmp2 activity reporter assay: schematic of the luciferase reporter system (left), representative fluorescence images, and statistical analysis of firefly luciferase activity by in vivo imaging system <t>(IVIS)</t> (a.u.: arbitrary units) (right, n = 3 per group). f. qPCR analysis of Bmp2 signaling pathway-related mRNA levels in MC3T3-E1 cells treated with the Bmp2 or D-Bmp2 protein (n = 3 per group). g, h. Western blot (g) and quantification of Bmp2 pathway-related protein expression in treated MC3T3-E1 cells (h) (n = 3 per group). i. Schematic of the HA-coated ELISA plate and HA-binding affinity assay (n = 3 per group). j. Tissue-specific binding assay of Bmp2 or D-Bmp2: Mouse muscle slices (left, scale bar: 100 μm) and undecalcified femur slices (right, scale bar: 200 μm) stained with AF647-conjugated anti-Flag antibodies. The data are presented as the means ± standard deviations (SDs). One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.
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optical microscope  (Olympus)
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Design and validation of bone-targeted Bmp2. a. Schematic representation of the Bmp2/D-Bmp2 plasmid constructs and the structure predicted by AlphaFold3. b. Immunofluorescence staining of HEK-293T cells transfected with the plasmids: phalloidin (green), DAPI (blue), and Alexa Fluor 647-conjugated anti-Flag antibodies (red). Scale bar: 10 μm. c. SDS-PAGE of purified proteins. d. Western blot validation of protein expression. e. Bmp2 activity reporter assay: schematic of the luciferase reporter system (left), representative fluorescence images, and statistical analysis of firefly luciferase activity by in vivo imaging system <t>(IVIS)</t> (a.u.: arbitrary units) (right, n = 3 per group). f. qPCR analysis of Bmp2 signaling pathway-related mRNA levels in MC3T3-E1 cells treated with the Bmp2 or D-Bmp2 protein (n = 3 per group). g, h. Western blot (g) and quantification of Bmp2 pathway-related protein expression in treated MC3T3-E1 cells (h) (n = 3 per group). i. Schematic of the HA-coated ELISA plate and HA-binding affinity assay (n = 3 per group). j. Tissue-specific binding assay of Bmp2 or D-Bmp2: Mouse muscle slices (left, scale bar: 100 μm) and undecalcified femur slices (right, scale bar: 200 μm) stained with AF647-conjugated anti-Flag antibodies. The data are presented as the means ± standard deviations (SDs). One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.
Optical Microscope, supplied by Olympus, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Design and validation of bone-targeted Bmp2. a. Schematic representation of the Bmp2/D-Bmp2 plasmid constructs and the structure predicted by AlphaFold3. b. Immunofluorescence staining of HEK-293T cells transfected with the plasmids: phalloidin (green), DAPI (blue), and Alexa Fluor 647-conjugated anti-Flag antibodies (red). Scale bar: 10 μm. c. SDS-PAGE of purified proteins. d. Western blot validation of protein expression. e. Bmp2 activity reporter assay: schematic of the luciferase reporter system (left), representative fluorescence images, and statistical analysis of firefly luciferase activity by in vivo imaging system (IVIS) (a.u.: arbitrary units) (right, n = 3 per group). f. qPCR analysis of Bmp2 signaling pathway-related mRNA levels in MC3T3-E1 cells treated with the Bmp2 or D-Bmp2 protein (n = 3 per group). g, h. Western blot (g) and quantification of Bmp2 pathway-related protein expression in treated MC3T3-E1 cells (h) (n = 3 per group). i. Schematic of the HA-coated ELISA plate and HA-binding affinity assay (n = 3 per group). j. Tissue-specific binding assay of Bmp2 or D-Bmp2: Mouse muscle slices (left, scale bar: 100 μm) and undecalcified femur slices (right, scale bar: 200 μm) stained with AF647-conjugated anti-Flag antibodies. The data are presented as the means ± standard deviations (SDs). One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Journal: Bioactive Materials

Article Title: Sustained release PLGA microspheres loaded with a bone-affinity Bmp2 enhance fracture healing and mitigate heterotopic ossification

doi: 10.1016/j.bioactmat.2026.02.050

Figure Lengend Snippet: Design and validation of bone-targeted Bmp2. a. Schematic representation of the Bmp2/D-Bmp2 plasmid constructs and the structure predicted by AlphaFold3. b. Immunofluorescence staining of HEK-293T cells transfected with the plasmids: phalloidin (green), DAPI (blue), and Alexa Fluor 647-conjugated anti-Flag antibodies (red). Scale bar: 10 μm. c. SDS-PAGE of purified proteins. d. Western blot validation of protein expression. e. Bmp2 activity reporter assay: schematic of the luciferase reporter system (left), representative fluorescence images, and statistical analysis of firefly luciferase activity by in vivo imaging system (IVIS) (a.u.: arbitrary units) (right, n = 3 per group). f. qPCR analysis of Bmp2 signaling pathway-related mRNA levels in MC3T3-E1 cells treated with the Bmp2 or D-Bmp2 protein (n = 3 per group). g, h. Western blot (g) and quantification of Bmp2 pathway-related protein expression in treated MC3T3-E1 cells (h) (n = 3 per group). i. Schematic of the HA-coated ELISA plate and HA-binding affinity assay (n = 3 per group). j. Tissue-specific binding assay of Bmp2 or D-Bmp2: Mouse muscle slices (left, scale bar: 100 μm) and undecalcified femur slices (right, scale bar: 200 μm) stained with AF647-conjugated anti-Flag antibodies. The data are presented as the means ± standard deviations (SDs). One-way ANOVA was used for multiple comparisons. Significance levels: ns (not significant), ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: After 24 h, the medium was changed to contain either Bmp2 or D-Bmp2 in DMEM, and the cells were further cultured for another 24 h. After the supernatant was removed, D-luciferin potassium salt (Beyotime, China) was added, and the firefly fluorescence signal was recorded using an in vivo imaging system (IVIS) (PerkinElmer, USA).

Techniques: Biomarker Discovery, Plasmid Preparation, Construct, Immunofluorescence, Staining, Transfection, SDS Page, Purification, Western Blot, Expressing, Activity Assay, Reporter Assay, Luciferase, Fluorescence, In Vivo Imaging, Enzyme-linked Immunosorbent Assay, Binding Assay