Journal: The Journal of investigative dermatology

Article Title: Increased expression of Wnt2 and SFRP4 in Tsk mouse skin: role of Wnt signaling in altered dermal fibrillin deposition and systemic sclerosis.

doi: 10.1038/sj.jid.5701101

Figure Lengend Snippet: Figure 5. Wnt3a stimulates Fbn-1 matrix formation. (a) MEFs were cultured for 10 days in the absence or presence of 100 ng/ml Wnt3a. Fbn-1 and fibronectin matrix (red) were analyzed by immunofluorescence. Nuclei were stained with Hoechst (blue). Bar ¼ 10 mm. The graph shows quantification of fibrils of the matrix by densitometric analysis of the fluorescent staining. (b) MEFs cultured for 16 hours in the absence or presence of 100 ng ml1 Wnt3a. Fbn-1 mRNA was analyzed by northern blot (left panels), and Fbn-1 and fibronectin protein levels in media by western blot (right panels). (c) Human dermal fibroblasts from normal or SSc skin were left untreated (left panel) or were treated with Wnt3a (right panel) for 10 days on chamber slides and then stained for Fbn-1 or fibronectin by immunofluorescence. Results are representative of three independent experiments performed on normal cells and SSc cells. (d) MEF-TE cells overexpressing MAGP-2 and tropoelastin-enhanced green fluorescent protein were cultured as in (a). Fbn-1 (red), MAGP-2 (red), elastic fibers (green), and fibronectin (red) were analyzed in parallel by immunofluorescence and enhanced green fluorescent protein fluorescence. Bar ¼ 10 mm. (e) MEFs were serum-starved for 3 hours and then treated for 1 hour with TGF-b1 (5 ng/ml) or Wnt3a (100 ng/ml). Total lysates were analyzed by western blot for phospho-Smad3 as described in Materials and Methods.

Article Snippet: Total lysates and secreted proteins were resolved on an SDS-PAGE gel, transferred to polyvinylidine difluoride membranes (Immobilon-P; Millipore) using standard methods, and then stained with Ponceau S. Membranes were then blocked in 5% milk or 5% BSA 0.05% Tween- trisbuffered saline for 1 hour, incubated overnight at 41C with monoclonal mouse anti-SFRP4 antibody (Berndt et al., 2003), polyclonal rabbit anti-Fbn-1 (Ab9543, gift from L. Sakai), anti- bovine fibronectin (Calbiochem, San Diego, CA), anti-phospho Smad3 (Rockland, Gilbertsville, PA), or anti-Smad2/3 (Upstate, Billerica, MA) and then with a horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Sigma, St. Louis, MO) or donkey anti- rabbit IgG (The Jackson Laboratory) for 1 hour.

Techniques: Cell Culture, Immunofluorescence, Staining, Northern Blot, Western Blot, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 1. Smad protein phosphorylation by TGF-1 stimulation in LX2, PSC, and MEF cells. LX2, PSC, and MEF cells were serum-starved overnight, then treated with different concentrations of TGF-1 for 45 min. Antibodies against p-Smad1/5 (Ser-463/465), p-Smad2 (Ser-465/467), and p-Smad3 (Ser-423/425) were used. Both Smad1/5 and Smad2/3 were phosphorylated by TGF-1 stimulation in all of the cells. An asterisk (*) indicates the nonspecific band.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 2. The effects of eliminating or overexpressing NRP-1 on Smad protein phosphorylation and the Smad target gene expression. A, eliminating NRP-1 up-regulated Smad1/5 phosphorylation and down-regulated Smad2/3 phosphorylation. NRP-1 was knocked down by siRNA in LX2 and PSC. For MEF cells, wt MEF, and NRP-1/ MEF cells were used. All the cells were serum-starved overnight, and then treated with 10 ng/ml TGF-1 for the indicated time. An asterisk (*) indicates the nonspecific band. B, overexpress NRP-1 in NRP-1/ MEF cells down-regulated Smad1/5 phosphorylation and up-regulated Smad2/3 phosphorylation. NRP-1/ MEF cells were infected with NRP-1 encoding retrovirus. LacZ retrovirus was used as control. After 36 h, cells were serum-starved overnight, and then treated with 10 ng/ml TGF-1 for the indicated time. C–E, effect of knocking down NRP-1 on the Smad proteins transcriptional activity in the cells measured by the luciferase promoter assay. 5 103/well LX2 cells were plated into a 96-well plate transfected with NRP-1 siRNA for 24–30 h, then serum-starved overnight. The cells were then transfected with the corresponding luciferase promoter plasmids together with pRL-TKRenilla luciferase plasmid astheinternalcontrol.Onehourafterthetransfection,TGF-1wasaddedtothecorrespondingwelltothefinalconcentrationof10ng/ml.Fireflyluciferaseand Renilla luciferase activities were performed. C, Luc-Id-1 promoter. D, Luc-ARE, co-transfected with FAST. E, Luc-SBE. F, knocking down NRP-1 affected the Smad protein responding gene expression. Cells were transfected with NRP-1 siRNA for 24–30 h in the complete medium, then serum-starved overnight. 10 ng/ml TGF-1 was added to the cells for 24 h. In response to TGF-1 stimulation, Id-1 protein (the Smad1-responding gene) expression was more highly up-regulated in NRP-1 knocked down cells than in control cells stimulated with TGF-1. In response to TGF-1 stimulation, -SMA and PAI-1 protein (the Smad3-responding gene) expression was more highly down-regulated in NRP-1 knocked down cells than in control cells stimulated with TGF-1.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics, Targeted Gene Expression, Infection, Control, Activity Assay, Luciferase, Promoter Assay, Transfection, Plasmid Preparation, Gene Expression

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 3. NRP-1 interacted with TGF- receptors. A, NRP-1 bound to TGF-RII. LX2 cells grown in the complete medium were made for cell lysate, and immunoprecipitation was performed using anti-NRP-1 antibody and West- ernblotforTGF-RII.ControlantibodywithcelllysateandNRP-1antibodywithoutcelllysatewereusedasnegative controls.B,TGF-1stimulationdidnotincreasetheassociationbetweenNRP-1andTGF-RII.LX2cellswereserum- starved overnight and stimulated with 10 ng/ml TGF-1 for 0, 15, and 45 min. The cell lysate was made, and immu- noprecipitation was performed using anti-NRP-1 antibody and Western blot for TGF-RII. C, siRNA knocked down TGF-RII eliminated the effect of NRP-1 on Smad protein phosphorylation. LX2 cells were transfected with control, NRP-1, and TGF-RII siRNA alone or combined, as indicated in the figure. 24–30 h later, cells were serum-starved overnight. 10 ng/ml TGF-1 was added to the indicated plates for 45 min, and the cell lysate was subjected to Western blot analysis. An asterisk (*) indicates the nonspecific band. D, TGF-RI (ALK5) inhibitors eliminated both Smad1/5 and Smad2/3 protein phosphorylation on TGF-1 stimulation. LX2 cells were serum-starved overnight. TGF-RI inhibitors SB431542 (10 M) and ALK5 Inhibitor (10 M) were added to the cells 30 min before TGF-1 stimulation. An asterisk (*) indicates the nonspecific band. E, NRP-1 associated with TGF-RI (ALK5) by TGF-1 stimulation. 293T cells were transfected with NRP-1 and Flag-ALK5-expressing plasmids. Cells were serum-starved overnight and stimulated with 10 ng/ml TGF-1 for 0, 5, and 15 min. The cell lysate was made, and immunoprecipi- tation was performed using anti-NRP-1 antibody and Western blot for Flag tag. F, knockdown TGF-RII eliminated theassociationbetweenNRP-1andTGF-RI(ALK5)inthepresentofTGF-stimulation.293Tcellsweretransfected with TGF-RII siRNA, and the following experiments were preformed as described in panel E.

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Transfection, Control, Expressing, FLAG-tag, Knockdown

Journal: Journal of Biological Chemistry

Article Title: Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

doi: 10.1074/jbc.m110.151696

Figure Lengend Snippet: FIGURE 8. The schematic illustration of NRP-1 function in regulating TGF- signaling. TGF- induced both Smad1/5/8 and Smad2/3 phosphorylation in the fibroblast cells. Without NRP-1 (left panel), Smad1/5/8 is more phosphorylated and Smad2/3 less phosphorylated, and the corresponding gene expression controlled by the Smad proteins (e.g. Smad1/Id-1, Smad3/a-SMA, PAI-1) caused the cell to enter a less activated state (more quiescent). With NRP-1 (right panel), Smad1/5/8 is less phosphorylated, and Smad2/3 is more phosphorylated, and the corresponding gene expression controlled by the Smad proteins caused the cell to enter a more activated state (less quiescent).

Article Snippet: Antibodies and Other Reagents—NRP-1, Id-1, T RII, and -actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); Smad2, Smad5, p-Smad1/5, p-Smad2, p-Smad3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA); -SMA was from Millipore (Billerica, MA); collagen I antibody was from Rockland Immunochemicals, Inc. (Gilbertsville, PA); PAI were fromNovus Bio. (Littleton, CO) TGF- 1 was from Biolegend (San Diego, CA); BMP9 was from R&D Systems (Minneapolis, MN); BMP2 and BMP4 were from Senway Biotech (San Diego,CA)ALK5 inhibitors SB431542 andALK5 inhibitors (2-(3- (6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) were from Stemgent (Cambridge, MA). siRNA and shRNA Transfection—siRNA for human NRP-1, T RII, and control were from Qiagen, Inc. (Valencia, CA). siRNA transfection was performed by Hiperfect (Qiagen) following the manufacturer’s instructions. shRNA for human NRP-1 and controls were from Open Biosystems (Huntsville, AL) and were prepared as previously described (18).

Techniques: Phospho-proteomics, Gene Expression

Journal: Molecular Medicine Reports

Article Title: Role and mechanism of tetrahedral DNA nanostructures in the repair of urethral injury in rats

doi: 10.3892/mmr.2026.13815

Figure Lengend Snippet: (A) Masson's trichrome staining and immunohistochemistry results. Masson's trichrome staining detected changes in collagen fibers in urethral tissues, with collagen fibers appearing blue and muscle fibers, fibrin and red blood cells appearing red. Immunohistochemistry staining showed the cell nuclei in blue and positive protein staining for the fibrosis markers α-SMA, TGF-β1, collagen I, collagen III and Smad3 in brown. Images are presented at a magnification of ×200. (B) Statistical analysis of Masson's trichrome staining and immunohistochemistry results. Staining results were analyzed with Image-Pro Plus software(version 6.0; Media Cybernetics, Inc.), followed by bar chart construction using GraphPad Prism. *P<0.05, **P<0.01 and ***P<0.001. (C) Western blot analysis results. Western blot analysis was used to detect the protein expression of fibrosis markers in urethral tissue. (D) Statistical analysis of western blotting results. Band densities were analyzed using Image-Pro Plus software(version 6.0; Media Cybernetics, Inc.), and bar charts were drawn with GraphPad Prism. *P<0.05, **P<0.01 and ***P<0.001. α-SMA, α-smooth muscle actin; TDN, tetrahedral DNA nanostructure.

Article Snippet: The membranes were blocked with 5% BSA for 1 h at room temperature, and incubated overnight at 4°C with the following primary antibodies: α-SMA (1:1,000; cat. no. AF1032; Affinity Biosciences), TGF-β1 (1:5,000; cat. no. 21898-1-AP; Proteintech Group, Inc.), collagen I (1:1,000; cat. no. AF7001; Affinity Biosciences), collagen III (1:1,000; cat. no. AF5457; Affinity Biosciences) and Smad3 (1:10,000; cat. no. 66516-1-Ig; Proteintech Group, Inc.), and β-actin (1:25,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.), which was used as a loading control.

Techniques: Staining, Immunohistochemistry, Software, Western Blot, Expressing

Journal: Molecular Cancer

Article Title: HDAC6 orchestrates metastatic and immunosuppressive programs in small cell lung cancer through S100A2-TGF-β/SMAD and CSF1R signaling

doi: 10.1186/s12943-025-02552-y

Figure Lengend Snippet: Acetylation status of S100A2 at lysine 27 site modulates the stability and nuclear translocation of downstream p-SMAD2/3 and SMAD2/3/4 complexes. a HDAC6-targeted deacetylation sites in S100A2. b Top 30 acetylated proteins and sites in NCI-H1688 HDAC6 WT vs OE visualized by radar plot. c-d FLAG-tagged S100A2 WT, K57R, K29R, K41R and K27R mutants were expressed in NCI-H1688 and SBC-2 cells by transient transfection. Anti-acetyl-lysine immuno-precipitates were western blotted with antibodies specific to FLAG. e–f Expression of p-SMAD2/3 downstream of S100A2 acetylation site mutants under different HDAC6 expression levels. g-h Stability of p-SMAD2/3 under different HDAC6 levels and S100A2 acetylation mutants after CHX treatment. i-j FLAG-tagged S100A2 NC, K27R, K27Q mutants and HA-tagged SMAD3 were expressed in NCI-H1688 and SBC-2 cells by transient transfection. Anti-HA and SMAD4 immuno-precipitates were western blotted with antibodies specific to FLAG. k-l Nuclear accumulation of p-SMAD2/3 under different HDAC6 levels and S100A2 acetylation mutants. m IF analysis of nuclear p-SMAD2 and SMAD4 levels under varying HDAC6 expression and S100A2 acetylation mutants in NCI-H1688 cell line. n Ch-IP assay was performed to validate changes in binding ability of SMAD3 to promoter of SNAI2 under SMAD3 WT or SMAD3 KO in HEK293T cell line. PC: positive control o Dual luciferase assay was performed to determine the promoter activity in HEK293T cell line

Article Snippet: Cells were probed with the following primary antibodies: anti-HDAC6 antibody (Proteintech Cat#12,834–1-AP, 1:100 dilution), anti-S100A2 antibody (Abcam Cat#ab109494, 1:100 dilution), anti-MMP9 antibody (Santacruz Cat#sc-21733, 1:100 dilution), anti-E-cadherin antibody (Santacruz Cat#sc-8426, 1:100 dilution), anti-p-SMAD2 (Ser465/Ser467) antibody (1:400 dilution, CST Cat#18,338) and anti-SMAD4 antibody (Proteintech Cat#10,231–1-AP, 1:500 dilution).

Techniques: Translocation Assay, Transfection, Western Blot, Expressing, Binding Assay, Positive Control, Luciferase, Activity Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Smad2 Protects against TGF-?/Smad3-Mediated Renal Fibrosis

doi: 10.1681/ASN.2009121244

Figure Lengend Snippet: Deletion of Smad2 enhances Smad3 phosphorylation in the UUO kidney and in vitro in response to TGF-β1. (A and B) Immunohistochemistry and Western blot analysis show that compared with the UUO kidney of Smad2ff mice, numbers of nucleated phospho-Smad3–positive cells and phospho-Smad3 protein are markedly increased in the UUO kidney of conditional Smad2 KO mice. (C) Knockdown of Smad2 from TECs (NRK52E) results in a significant increase in phospho-Smad3 at 30 minutes (peak time) after TGF-β1 stimulation. (D) Western blot analysis shows that compared with Smad2 WT MEFs, addition of TGF-β1 (2 ng/ml) largely enhances Smad3 phosphorylation in Smad2 KO MEFs in a time-dependent manner when compared with the Smad2 WT MEFs. Data are means ± SEM for groups of eight mice in vivo and four independent experiments in vitro. *P < 0.05, **P < 0.01, ***P < 0.001 versus time (dosage) 0 or normal; #P < 0.05, ##P < 0.01, ###P < 0.001 versus Smad2WT MEFs or injured Smad2ff (UUO) mice.

Article Snippet: 36 The antibodies used in this study included phospho-Smad3 (Rockland Immunochemicals, Gilbertsville, PA), CTGF, TGF-β1 (Santa Cruz Biotechnology), and collagen I and collagen III (Southern Tech, Birmingham, AL).

Techniques: In Vitro, Immunohistochemistry, Western Blot, In Vivo

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Smad2 Protects against TGF-?/Smad3-Mediated Renal Fibrosis

doi: 10.1681/ASN.2009121244

Figure Lengend Snippet: Deletion of Smad2 enhances Smad3 signaling in MEFs. (A) Immunofluorescence detects that MEFs lacking Smad2 substantially enhance phosphorylated Smad3 nuclear translocation at 30 minutes after TGF-β1 (2 ng/ml) stimulation when compared with the Smad2 WT MEFs. (B) Quantitative analysis of phospho-Smad3 nuclear translocation in response to TGF-β1 (2 ng/ml). (C) Smad3 promoter activity assay. (D) ChIP assay for binding of Smad3 to the COL1A2 promoter. (E) Quantitative real-time PCR analysis of the binding of Smad3 to COL1A2. Data are means ± SEM for four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus time 0 or empty vector control; #P < 0.05, ##P < 0.01, ###P < 0.001 versus Smad2 WT MEFs.

Article Snippet: 36 The antibodies used in this study included phospho-Smad3 (Rockland Immunochemicals, Gilbertsville, PA), CTGF, TGF-β1 (Santa Cruz Biotechnology), and collagen I and collagen III (Southern Tech, Birmingham, AL).

Techniques: Immunofluorescence, Translocation Assay, Activity Assay, Binding Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Smad2 Protects against TGF-?/Smad3-Mediated Renal Fibrosis

doi: 10.1681/ASN.2009121244

Figure Lengend Snippet: Overexpression of Smad2 attenuates the fibrotic effect of TGF-β1 in TECs. (A) Characterization of Smad2-overexpressing TECs. (B) Western blot analysis shows that compared with empty vector control (VC), overexpression of Smad2 (S2Over) inhibits TGF-β1–induced (2 ng/ml) phosphorylation of Smad3 in TECs (NRK52E). (C) Real-time PCR shows that overexpression of Smad2 in TECs attenuates the fibrosis response to TGF-β1 (2 ng/ml, 3 hours), including a significant inhibition of TGF-β1, CTGF, collagens I and III, and TIMP mRNA expression, while increasing MMP-2 expression. Data are means ± SEM for four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus time 0 or empty vector control; #P < 0.05, ##P < 0.01, ###P < 0.001 versus empty vector control (VC).

Article Snippet: 36 The antibodies used in this study included phospho-Smad3 (Rockland Immunochemicals, Gilbertsville, PA), CTGF, TGF-β1 (Santa Cruz Biotechnology), and collagen I and collagen III (Southern Tech, Birmingham, AL).

Techniques: Over Expression, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Inhibition, Expressing

Journal: eLife

Article Title: The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates Drosophila wing patterning and growth

doi: 10.7554/eLife.73357

Figure Lengend Snippet: ( A ) Co-immunoprecipitation of Nord with the BMP ligands Dpp and Glass-bottom boat (Gbb). Medium from S2 cells transfected for expression of GFP-tagged Nord were mixed with medium from cells expressing FLAG-tagged Dpp and HA-tagged Gbb alone or in combination, followed by incubation with anti-FLAG or anti-HA antibody-coupled beads overnight at 4°C. Precipitated proteins were analyzed by Western blotting with indicated antibodies. Nord was immunoprecipitated with Dpp and, to a lesser extent, with Gbb. The amount of immunoprecipitated Nord was increased when Dpp and Gbb were co-transfected. ( B ) S2 cells were transfected for expression of FLAG-tagged Dpp or Gbb with or without GFP-tagged Nord (source cell). Both cell lysate and conditioned medium from the source cells were collected and followed by Western blot analysis. Loading was controlled by probing the blot for tubulin. The amount of Dpp or Dpp-Gbb ligands released into the medium was reduced when Nord was co-expressed in the source cells. ( C ) Comparison of BMP signaling activities of conditioned media in a cell-based signaling assay. After incubating the conditioned media collected in ( B ) with S2 cells stably expressing the FLAG-Mad transgene (Mad-S2, responding cell) for 1 hr at room temperature, the responding cells were washed and lysed. The lysates were probed with anti-pMad and anti-FLAG antibodies to detect both the phosphorylated Mad and total Mad protein, respectively. ( D, E ) Quantification of the Western blot data in panels ( B ) and ( C ). The levels of the secreted BMP ligands from the medium (anti-FLAG in panel B ), phosphorylated Mad (anti-pMad in panel C ), and total Mad (anti-FLAG in panel C ) were measured based on the band intensity. The signaling activity from an equal volume of conditioned medium was determined by the ratio of pMad and the corresponding total Mad (pMad/Mad) in panel ( C ), which was then normalized to the condition without the exogenous Nord-GFP to calculate a relative signaling activity ( D ), or normalized to the secreted ligand amount to calculate the relative ligand activity ([pMad/Mad]/BMPs) ( E ). ( F ) Mad-S2 cells were treated with a recombinant Dpp peptide (rDpp) in the absence or presence of conditioned medium containing raising levels of Nord for 1 hr at room temperature, the responding cells were washed and lysed. The lysates were probed with anti-pMad and anti-FLAG antibodies to detect both the phosphorylated Mad and total Mad protein, respectively. ( G ) Quantification of the Western blot data in panel ( F ). The phosphorylated Mad (anti-pMad) and total Mad (anti-FLAG) levels were measured based on the band intensity. The signaling activity from each conditioned medium was determined by the ratio of pMad and the corresponding total Mad (pMad/Mad), which was then normalized to the condition with 2 nM Dpp but no additional Nord. Panel ( F ) is representative of n = 3 independent experiments. Each point shows the mean ± SD. One-way ANOVA test with Tukey’s multiple comparison was used for statistical analysis, and a significant difference was considered by *p<0.05. ( H ) Mad-S2 cells were transiently transfected for expression of GFP or Nord-GFP. 48 hr after transfection, the cells were treated with recombinant Dpp peptides for 1 hr. Upon treatment, the cells were washed, fixed, and stained by anti-FLAG to detect total Mad and anti-pMad to detect phosphorylated Mad. The average pMad levels were measured and normalized to the total Mad levels, and then plotted against different Dpp concentrations (0–5 nM). Each point shows the mean ± SD, n > 10. au, arbitrary units. The unpaired two-tailed t -test was used for statistical analysis. ***p<0.001, ****p<0.0001. ( I ) Representative images of Mad-S2 cells treated by 0.625 nM Dpp. Scale bar, 10 μm. Figure 8—source data 1. Uncropped Western blot for .

Article Snippet: Recombinant Dpp (159-DP-020, R&D Systems) was diluted in the culture medium according to the manufacturer’s recommendations.

Techniques: Immunoprecipitation, Transfection, Expressing, Incubation, Western Blot, Comparison, Stable Transfection, Activity Assay, Recombinant, Staining, Two Tailed Test

Journal: eLife

Article Title: The NDNF-like factor Nord is a Hedgehog-induced extracellular BMP modulator that regulates Drosophila wing patterning and growth

doi: 10.7554/eLife.73357

Figure Lengend Snippet:

Article Snippet: Recombinant Dpp (159-DP-020, R&D Systems) was diluted in the culture medium according to the manufacturer’s recommendations.

Techniques: Magnetic Beads, Modification, Transfection, Electron Microscopy, Recombinant, Reverse Transcription, SYBR Green Assay, Sequencing, Clone Assay, Plasmid Preparation, In Situ, CRISPR, Software