Dapi, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Modulation of occluding junctions alters the hematopoietic niche to trigger immune activation"
Article Title: Modulation of occluding junctions alters the hematopoietic niche to trigger immune activation
Figure Legend Snippet: Bacterial infection induces changes in the prohemocyte microenvironment. ( a–b’ ) PSC cell numbers increase upon infection with ( a ’’,a’’’ ) B. subtilis and ( b,b’ ) E. coli as compared to ( a,a’ ) sucrose treated control. ( b’’ ) Quantitation of PSC cell numbers upon infection. 70 kDa dextran (Red) can access the PSC cells upon infection with either B. subtilis ( d–d’’’ ) or E. coli ( e–e’’’ ) as compared to the sucrose control ( c–c’’’ ). ( f ) Quantitation of 70 kDa dye influx upon bacterial infection. Circulating hemocytes are activated and show increased phalloidin (Green) positive filopodial extensions upon infection with B. subtilis ( g’,h’ ) or E. coli ( g’’,h’’ ) as compared to the sucrose control ( g,h ). ( i ) Flies having prior exposure to E. coli in larval stages ( L ) have better survival ability upon subsequent infection in adult stages ( A ) as compared to the respective controls. ( j–l’’ ) Coracle (Red) expression in the PSC is down-regulated upon systemic infection with B. subtilis ( k–k’’ ) or E. coli ( l–l’’ ) as compared to sucrose control ( j–j’’ ). ( m–n’ ) NrxIV::GFP expression (Green) in the PSC marked with Antp (Red) is down-regulated upon systemic infection with E. coli ( n–n’ ) as compared to sucrose control ( m–m’ ). ( o–r’’ ) Eater-dsRed (Red) positive plasmatocytes or Hnt (Red) positive crystal cells are increased upon systemic infection with B. subtilis ( p–p’’,r’ ) or E. coli ( q–q’’,r’’ ) as compared to sucrose control ( o–o’’,r ). ( s–t ) Survival plots for flies systemically infected with B. subtilis or E. coli over time in control versus PSC specific Cora knockdown flies. ( a,a’’,b,c–e’’, j–l’’ and o–r’’ ) PSC is labeled in green with Collier-GFP (UAS-GFP driven by collier-Gal4 ) and Antennapedia (Red:a,a’’,b, m-n’; white:a’,a’’’,b’). ( i ) Suc indicates sucrose control while infection indicates bacterial infection for the survival analysis. Plasmatocytes are labeled with Eater-dsRed (Red:o-q’’), crystal cells with Hnt (Red: r-r’’). Nuclei are labeled with DAPI (Blue). *** indicates p
Techniques Used: Infection, Quantitation Assay, Expressing, Labeling
2) Product Images from "The Dystrophin Glycoprotein Complex Regulates the Epigenetic Activation of Muscle Stem Cell Commitment"
Article Title: The Dystrophin Glycoprotein Complex Regulates the Epigenetic Activation of Muscle Stem Cell Commitment
Journal: Cell stem cell
Figure Legend Snippet: p38γ MAPK Phosphorylates Carm1 on Ser 572 (A) Coomassie blue staining of Carm1 treated with activated p38γ (+MKK6EE) or non-activated p38γ (+pcDNA3) for LC-MS/MS analysis. Asterisks denote Carm1 and arrowheads denote p38γ kinase. (B) MS/MS spectrum of Carm1 peptide containing phosphorylated S572. Matched b and y ions and the position of oxidations (OX) and phosphorylations (PH) are indicated. (C) Diagram of Carm1 ΔE15 protein with indicated phosphorylation site at S572. (D) Multiple sequence alignment of the last 35 amino acids of Carm1 in 5 different vertebrate species. S572 is marked in red. An asterisk (*) represents identical amino acids, a colon (:) represents highly conserved amino acids, and a period (.) represents weakly conserved amino acids. (E) In vitro kinase assays between active p38γ and wild type or S572A mutant Carm1. The asterisk denotes phosphorylated Carm1 and the arrowhead denotes active p38γ kinase. (F) Carm1:p-S/T-P PLA (red) performed on satellite cells cultured on myofibers isolated from p38γ +/+ or p38γ fl/fl mice. Satellite cells are marked by expression of Syndecan-4 (Sdc4, green) and nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm. .
Techniques Used: Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, In Vitro, Mutagenesis, Proximity Ligation Assay, Cell Culture, Isolation, Mouse Assay, Expressing
Figure Legend Snippet: p38γ Negatively Regulates Asymmetric Satellite Stem Cell Division and is Required for Symmetric Self-Renewal (A) Schematic of siRNA-treated myofiber culture experiments performed in Figure 4. (EDL, extensor digitorum longus . PLA, proximity ligation assay. IF, immunofluorescence). (B) Carm1:Pax7 PLA (red) performed on satellite cells cultured for 42h on myofibers that were treated with indicated siRNAs. Satellite cells are marked by expression of Sdc4 (green) and nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm. (C) Quantification of PLA signal from (B), represented as the mean (n = 39 si-Ctrl, n = 54 si-p38γ, n = 47 siCarm1 cells from 3 mice) ± SEM (*p ≤ 0.05). The PLA was quantified by counting the number of nuclear PLA puncta for each satellite cell. (D) Immunofluorescence of satellite cells cultured for 42h on myofibers that were treated with either control (si-Ctrl) or p38γ (si-p38γ) siRNA. Cells were immunostained for Sdc4 (green), and Myf5 (red). Nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm. (E) Quantification of mean fluorescence intensity of Myf5 staining from (D), represented as the mean (n = 67 si-Ctrl, n = 69 si-p38γ cells from 3 mice) ± SEM (***p ≤ 0.001). (F) Quantification of the number of asymmetric satellite stem cell (Pax7 + /YFP − ) divisions per myofiber, represented as the mean (n = 9 mice) ± SEM (**p ≤ 0.01. NS, not significant). (G) Quantification of the number of symmetric satellite stem cell (Pax7 + /YFP − ) divisions per myofiber, represented as the mean (n = 9 mice) ± SEM (*p ≤ 0.05. NS, not significant). (H) Quantification of the number of committed satellite progenitor cell (Pax7 + /YFP + ) divisions per myofiber, represented as the mean (n = 9 mice) ± SEM (NS, not significant). .
Techniques Used: Proximity Ligation Assay, Immunofluorescence, Cell Culture, Expressing, Mouse Assay, Fluorescence, Staining
Figure Legend Snippet: p38γ/Carm1 interacts with β1-Syntrophin in Satellite Cells (A) Immunofluorescence of satellite cells cultured for 36h on myofibers isolated from wild type (WT) or mdx mice. Cells were immunostained for α7 integrin (white), β1-syntrophin (green), and p38γ (red). Nuclei were counterstained with Hoechst (blue). Scale bar represents 10 μm. (B, C, and D) PLA with indicated antibodies (red) performed on satellite cells cultured for 36h on single EDL myofibers. Satellite cells were identified by their expression of α7 integrin (white) and nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm. (E, and F) PLA with indicated antibodies (red) performed on satellite cells cultured for 36h on single myofibers isolated from either WT or mdx mice. Satellite cells were identified by their expression of Sdc4 (green) and nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm. (G) Quantification of PLA in (F), represented as the relative mean (n = 21 WT, n = 26 mdx cells from 3 mice) ± SEM (**p ≤ 0.01). The PLA was quantified by measuring mean fluorescence intensity of the PLA signal for each satellite cell. .
Techniques Used: Immunofluorescence, Cell Culture, Isolation, Mouse Assay, Proximity Ligation Assay, Expressing, Fluorescence
Figure Legend Snippet: Carm1 is Directly Phosphorylated by p38γ MAPK (A) In vitro kinase assays between GST-Carm1 with either FLAG-p38γ or FLAG-p38α kinases. p38 kinases were activated by co-expression of MKK6EE, as indicated. The asterisk denotes phosphorylated Carm1. Arrowheads denote p38γ (γ) and p38α (α) kinases. (B) In vitro kinase assays between GST-Carm1 with either wild type Myc-p38γ or kinase-inactive Myc-p38γ AF. The asterisk denotes phosphorylated Carm1 and the arrowhead denotes active Myc-p38γ kinase. (C) HEK 293T cells were transfected with FLAG-Carm1 and either Myc-p38γ, Myc-p38γ AF, or pcDNA3. Immunoprecipitation (IP) and immunoblotting were performed with the indicated antibodies. (D) Carm1:p38γ PLA (red) performed on satellite cells cultured on myofibers. Satellite cells are marked by expression of Pax7 (green) and nuclei were counterstained with DAPI (blue). Scale bar represents 20 μm. .
Techniques Used: In Vitro, Expressing, Transfection, Immunoprecipitation, Proximity Ligation Assay, Cell Culture
3) Product Images from "New Roles for Cyclin E in Megakaryocytic Polyploidization *"
Article Title: New Roles for Cyclin E in Megakaryocytic Polyploidization *
Journal: The Journal of Biological Chemistry
Figure Legend Snippet: Immunofluorescence detection of hCyclin E in MKs. Immunofluorescence microscopy confirms human cyclin E expression in bone marrow-derived MKs of the transgenic mouse model (line 11 is shown) or wild-type matching controls ( Wt ). Human cyclin E (hcyclin E) antibody was used, followed by a secondary staining with goat anti- mouse Alexa-488 as described under “Experimental Procedures.” HeLa cells were used as a positive control. Cells were also stained with DAPI ( blue ) to visualize DNA using fluorescent microscopy. Large polyploid MKs are readily identifiable.
Techniques Used: Immunofluorescence, Microscopy, Expressing, Derivative Assay, Transgenic Assay, Staining, Positive Control
4) Product Images from "Peroxisome proliferator-activated receptor ? activation promotes myonuclear accretion in skeletal muscle of adult and aged mice"
Article Title: Peroxisome proliferator-activated receptor ? activation promotes myonuclear accretion in skeletal muscle of adult and aged mice
Journal: Pflugers Archiv
Figure Legend Snippet: GW0742-promoted myonuclear remodelling does not require cell division in tibialis anterior. Mice were injected with BrdU and or not with GW0742. Duodenum ( a , b ) or TLA muscle ( c – f ) harvested from 24 h ( b ) or 48 h ( a , c – f ) post GW0742 treatment, were either frozen in tissue-embedding medium or fixed, dehydrated and embedded in paraffin. Frozen sections on slides were stained with anti-BrdU antibody coupled to fluorescein ( a , c , e ) and mounted using Vectashield containing DAPI as described in “ Materials and methods ”. Positive cells are detected in blood vessels, and very few myonuclei are labelled ( c , e ). Arrowheads indicate myofibres with central nucleus ( c , e ). Paraffin sections were stained with anti-BrdU antibody ( b , d , f ) as described in “ Materials and methods ”, and nuclei were counterstained with haematoxylin. Very few myonuclei are labelled ( d , and arrow in f ); on the contrary, a large number of myofibres with one or more central nuclei are visible ( d , e , f ). Note the increased BrdU labelling in the duodenum sections between 24 h ( b ) and 48 h ( a ) of the BrdU pulse. Scale bar, 50 μm
Techniques Used: Mouse Assay, Injection, Staining
5) Product Images from "Enteric Glia Regulate Gastrointestinal Motility but are not Required for Maintenance of the Epithelium in Mice"
Article Title: Enteric Glia Regulate Gastrointestinal Motility but are not Required for Maintenance of the Epithelium in Mice
Figure Legend Snippet: Gfap HSV-TK mice exhibit transgene expression throughout the intestine and signs of off-target injury to the epithelium (A, D) Immunostaining for S100β and HSV-TK shows that only a subset of myenteric glia expresses TK (arrows) in the ileum and colon of untreated GFAP HSV-TK mice. Intramuscular glia are S100β-immunoreactive but none express TK. (B–F) Valganciclovir (VGCV) treatment eliminates both GFAP + (compare B and C) and S100β + (compare E and F) enteric glia in the small and large intestine of GFAP HSV-TK mice. NF-H immunostaining highlights nerve fiber bundles in the myenteric plexus. (G) Serum FITC levels 4 hours after oral gavage of 478Da fluorescein conjugate into TK − and TK + mice treated with VGCV shows increased macromolecular intestinal permeability in TK + mice. (H–I) Immunoreactivity of the apoptotic marker, phosphorylated histone 2AX (pH2AX) in cross-sections of ilea from TK − (H) and TK + (I) mice treated with VGCV. Nuclei counterstained with DAPI. VGCV treatment increased apoptosis primarily in crypt cells, but also in the walls of the villi and in the lamina propria of the TK + mouse. (J) The ultrastructure of enterocytes in the small intestine of a control TK − mouse treated with VGCV appears normal. The microvillus (mv) border was intact, and their core of actin filaments formed a terminal web and inserted into the adherens junction component of junctional complexes (arrow). (K) The ultrastructure of some enterocytes in the small intestines of VGCV-treated TK + mice was abnormal. The microvilli were disorganized, lacked microfilament cores, and displayed variable diameters suggesting swelling. The area of the terminal web was expanded and appeared as an amorphous electron-dense mat. Occasionally, adherens junctions were disrupted although tight junctions remained intact. In rare cells, the underlying cytoplasm appeared to push the microvillus border aside and protrude into the lumen (see inset). In these regions, tight junctions were disrupted, opening a channel to the intercellular space. (L) In an evident later stage of abnormality in a VGCV-treated TK + mouse, the cytoplasm of some enterocytes was electron dense, the terminal web (tw) region was expanded, and the mitochondrial matrix was vacuolated. Scale bars A–I=50μm; J–L=500nm
Techniques Used: Mouse Assay, Expressing, Immunostaining, Permeability, Marker
6) Product Images from "Landscape of monoallelic DNA accessibility in mouse embryonic stem cells and neural progenitor cells"
Article Title: Landscape of monoallelic DNA accessibility in mouse embryonic stem cells and neural progenitor cells
Journal: Nature genetics
Figure Legend Snippet: RAMA elements are stable through mitosis and over many passages in the NPC state. ( a ) Experimental setup for allele-specific (AS) ATAC–seq across passages. Three NPC lines (XX4, XX2 and XY14) were cultured for five and ten additional passages (PX + 5 and PX + 10, respectively) after the initial ATAC–seq experiment (PX + 0). ( b ) ATAC–seq d scores at passage 0 versus passage 10 for promoter RAMA elements in NPC clones XX2, XX4 and XY14. ( c ) ATAC–seq d scores at passage 0 versus passage 10 for distal RAMA elements in NPC clones XX2, XX4 and XY14. ( d ) Experimental setup for mitotic ATAC–seq. NPC clone XX1 was blocked in M phase, and mitotic cells were then collected by shaking. The image shows mitotic NPCs stained with DAPI for mitotic chromosomes and antibody to H3S10ph, a marker of prometaphase cells (40× magnification). ( e ) ATAC–seq d scores for RAMA elements in clone XX1 in mitotic versus asynchronous cells. Monoallelic RAMA elements were included if there were ≥20 allele-informative reads under the peak in both mitotic and asynchronous ATAC–seq data. ( f ) Ratio of ATAC–seq read peaks in asynchronous/mitotic cells for promoter elements (
Techniques Used: Cell Culture, Clone Assay, Staining, Marker
7) Product Images from "Genetic Variants on Chromosome 1q41 Influence Ocular Axial Length and High Myopia"
Article Title: Genetic Variants on Chromosome 1q41 Influence Ocular Axial Length and High Myopia
Journal: PLoS Genetics
Figure Legend Snippet: Immunofluorescent labeling. Immunofluorescent labeling of (A) ZC3H11A (B) SLC30A10 and (C) LYPLAL1 in mouse retina, retinal pigment epithelium and sclera in induced myopic eyes, fellow eyes and independent control eyes. The neural retina (retina), retinal pigment epithelium (PRE) and scleral cells were immunolabeled with the polycolonal antibodies against ZC3H11A , SLC30A10 and LYPLAL1 and were co-labeled with 4′,6-diamidino-2-phenylindole (DAPI). Negative controls were devoid of a fluorescence signal, treated with the secondary antibody alone and DAPI. No immunostaining was observed in the negative controls. Scale bar represents 50 µM and magnification is 200×. The florescence intensity labeled of the green color shows the localization of proteins and blue color indicates the nuclei that were stained with DAPI. Expression of the proteins had a trend in abundance similarly to that of their mRNA levels as depicted in Figure 4 . Lower level of expression was determined for ZC3H11A in all tissues for myopic mice. Similarly significant reduction was shown in the expression of SLC30A10 in retina and RPE while higher level of expression was found in myopic sclera. LYPLAL1 showed higher level of expression in the retina and RPE tissue but reduced expression in the sclera in myopic mice. The following abbreviations represent the retinal layers: nerve fibre layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), photo receptor layer (PRL) and retinal pigment epithelium (RPE).
Techniques Used: Labeling, Immunolabeling, Fluorescence, Immunostaining, Staining, Expressing, Mouse Assay
8) Product Images from "Therapeutic Effects of Insulin-Producing Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Type 1 Diabetes Mouse Model"
Article Title: Therapeutic Effects of Insulin-Producing Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Type 1 Diabetes Mouse Model
Journal: International Journal of Molecular Sciences
Figure Legend Snippet: Trans-differentiation of mesenchymal stem cells. ( a ) The trans-differentiation protocol for obtaining pancreatic β-cells. Key transcription factor hierarchy during pancreas development. ( b ) Immunofluorescent detection of MAFA (Red) and insulin (green), as well as 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining (blue) in cultured hUC-MSCs and hAD-MSCs during step 3. The merged image of MAFA and insulin is shown. Images of all fluorescently stained cells were acquired using a ×100 objective, and representative images are shown. ( c ) Immunofluorescent detection of C-peptide (red), insulin (green), as well as DAPI nuclear staining (blue) in cultured hUC-MSCs and hAD-MSCs during step 3. The merged image of C-peptide and insulin-producing β-cells is shown. Images of all fluorescently stained cells were obtained using a ×100 objective, and representative images are shown. Differentiation of hUC-MSCs and hAD-MSCs into IPCs. ( d ) RT-PCR analysis of pancreatic cell marker expression in IPCs differentiated from hUC-MSCs and hAD-MSCs. Marker genes include neurogenin-3 ( NGN3 ), neuronal differentiation ( NEUROD ), INS , MAF BZIP transcription factor A ( MAFA ), glucose transporter 2 ( GLUT2 ), somatostatin ( SST ), normalized to the glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) gene. ( e ) Quantitative analysis of mRNA expression during IPC differentiation from hUC-IPCs and hAD-IPCs during step 3. Results are presented as the mean ± standard error of the mean. Significant differences between hAD-IPCs and hUC-IPCs are noted at ** p
Techniques Used: Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Marker, Expressing
Figure Legend Snippet: Application of cell therapy agents in a T1D mouse model. All fluorescently stained cells were observed under a ( a ) ×40 objective and a ( b ) ×50 objective, and representative images are shown. The white arrows indicate merged cells. Abbreviations: G, group; DAPI, 4′,6-diamidino-2-phenylindole.
Techniques Used: Staining
9) Product Images from "Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes"
Article Title: Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes
Journal: Nature Communications
Figure Legend Snippet: Tubule characterization by immunofluorescent staining. a 3D reconstruction of a confocal z -stack showing tubular morphology with a lumen. White arrows indicate the apical ( A ) and basal ( B ) sides. The tube is stained for tight junctions (ZO-1 in red ) and brush borders (ezrin in green ). b Max projection and c vertical cross-section of the tubular structure in a ; d , e zoom of the epithelial layer at the bottom of the tube exhibiting d tight junctions (ZO-1 in red ) and brush borders (ezrin in green ), and e acetylated tubulin ( green ) and occluding ( red ). f Phase-contrast image showing dome formation. g Zoom of a z -slice of the tube in a of the cell layer on top of the phaseguide showing apical positioning of ezrin, indicating polarization of the tube ( white arrow indicates basal side B). h Expression of glucose and MRP2 transporters, respectively stained with Glut-2 in red and MRP2 stain in green . Both Glut-2 and MRP2 show significantly higher signal against the collagen gel compared to the regions that are not exposed to the collagen, indicating increased expression levels. Both stains clearly stain the apical side of the tube. For z -slices above the phaseguide at a higher magnification see Supplementary Fig. 2b . i ErbB1 ( red ) and acetylated tubulin ( green ) expression. ErbB1 expression levels appear higher against the collagen. j Co-staining of Glut-2 transporter and ErbB2 receptor; both stains show higher signal levels against the collagen gel. ErbB2 is primarily expressed pericellularly (see also Supplementary Fig. 2d for a zoom)). All tubes are fixed after 4 days in culture. Nuclei are stained blue with Draq5 ( a – c , g – j ) and DAPI ( d , e ). Scale bars in white are 100 µm with the exception of d , e , f , and g , where they are 50 µm. Z -slices just above the phaseguide at higher magnification of the images g – j are available in Supplementary Fig. 2 . All images are representative of at least three biological and at least three technical replicates
Techniques Used: Staining, Expressing
10) Product Images from "In mouse embryonic fibroblasts, neither caspase-8 nor cellular FLICE-inhibitory protein (FLIP) is necessary for TNF to activate NF-κB, but caspase-8 is required for TNF to cause cell death, and induction of FLIP by NF-κB is required to prevent it"
Article Title: In mouse embryonic fibroblasts, neither caspase-8 nor cellular FLICE-inhibitory protein (FLIP) is necessary for TNF to activate NF-κB, but caspase-8 is required for TNF to cause cell death, and induction of FLIP by NF-κB is required to prevent it
Journal: Cell Death and Differentiation
Figure Legend Snippet: TNF-induced nuclear translocation of p65/RelA NF- κ B is not impaired in caspase-8 −/− or FLIP −/− MEFs. ( a ) WT MEFs, caspase-8 −/− , FLIP −/− , and NEMO −/− MEFs were cultured with or without 70 ng/ml TNF for 20 min and stained with an anti-p65 antibody (green) and DAPI (blue), and visualized by immunofluorescence microscopy. Nuclear translocation of p65/RelA occurred within 20 min of TNF treatment, except in NEMO −/− MEFs, which were used as control to show defective p65/RelA translocation. ( b ) Absence of caspase-8 and FLIP in caspase-8 −/− and FLIP −/− MEFs. Cell lysates from control and gene-deleted MEFs were probed for caspase-8 or FLIP. β -Actin was used as a loading control. ( c ) TNF-induced I κ B degradation is not impaired in caspase-8 −/− and FLIP −/− MEFs. Caspase-8 lox/lox , caspase-8 −/− , FLIP +/− , and FLIP −/− MEFs were treated with 100 ng/ml TNF for the times indicated. Cell lysates were probed for I κ B α and β -actin was used as a loading control
Techniques Used: Translocation Assay, Cell Culture, Staining, Immunofluorescence, Microscopy
11) Product Images from "Integration of Multiple Signaling Regulates through Apoptosis the Differential Osteogenic Potential of Neural Crest-Derived and Mesoderm-Derived Osteoblasts"
Article Title: Integration of Multiple Signaling Regulates through Apoptosis the Differential Osteogenic Potential of Neural Crest-Derived and Mesoderm-Derived Osteoblasts
Journal: PLoS ONE
Figure Legend Snippet: Differential activation of endogenous TGF-β and BMP signaling between FOB and POb cells. (A) Immunoblotting analysis using specific anti-phosphoSmad-2 and phoshoSmad-1/5 antibodies shows a more intense phosphorylation of Smad-2 in POb than FOb cells. In contrast, analysis with anti-phoshoSmad-1/5 antibody reveals a stronger staining in FOb. To assess for the total amount of endogenous Smad-2 and Smad-1/5 and to control for equal loading and transfer of the samples membranes were reprobed with anti-Smad-2, Smad-1/5 and anti-α-Tubulin antibodies. Histogram below represents quantification of phosphorylated Smad-2 and Smad-1/5 proteins obtained by Image J program. The relative intensity of each band was normalized to their respective α-Tubulin loading controls. (B) immunofluorescent staining using anti-phosphoSmads antibodies as above confirms the results obtained by immunoblotting analysis. Immunofluorescent staining using anti-phosphoBcl-2 antibody detects higher levels of the anti-apoptotic protein Bcl-2 in FOb compared to POb. Dapi nuclear counterstaining. (C) Immunoblotting analysis performed as above (A) showing that the differential activation of the two signaling pathways observed between FOb and POb cells is maintained throughout their osteogenic differentiation.
Techniques Used: Activation Assay, Staining