alk3 fc  (R&D Systems)

Journal: Advanced Science

Article Title: Autocrine GDF10 Inhibits Hepatic Stellate Cell Activation via BMPR2/ALK3 Receptor to Prevent Liver Fibrosis

doi: 10.1002/advs.202500616

Figure Lengend Snippet: Characterization of the interaction between GDF10 and BMPR2/ALK3. A) Percentage of expression + binding + transfectants of HEK293 cells expressing a single TGF‐β superfamily receptor incubated with GDF10‐Fc. B) Percentage of expression + binding + transfectants of HEK293 cells expressing BMPR2 and type I TGF‐β receptor incubated with GDF10‐Fc. C) HEK293 cells were transfected to express BMPR2 and ALK3 and then incubated with GDF10‐Fc, scale bars, 50 µm. D) Association of BMPR2 with ALK3. HEK293 cells were transfected to express BMPR2‐FLAG and ALK3‐HIS. E) The overall structure of mature GDF10 with the extracellular domains (ECD) of mouse BMPR2 and ALK3 (GDF10: blue‐purple, BMPR2: pink‐green, ALK3: pink). F) Western blot analysis of cell lysates from HEK293 cells transfected to express C‐terminal FLAG‐tagged BMPR2 and FLAG‐tagged ALK3 incubated with GDF10‐Fc and then immunoprecipitated with anti‐FLAG antibody. G) Biacore sensorgrams and binding kinetics were determined by SPR spectroscopy for GDF10 with the ECD of mouse BMPR2 and ALK3. H) qPCR analysis of the expression Bmpr2 and Alk3 in mouse LSECs, MACs, HSCs, and HCs ( n = 6). I) Cell type enrichment of BMPR2 and ALK3 in human liver (Human Protein Atlas database).

Article Snippet: For Western blot analysis, the following primary antibodies were employed: β‐Actin (AC026; ABclonal), GDF10 (ab235005; Abcam), ACTA2 (A17910; ABclonal), COL1A1 (A22089; ABclonal), phospho‐SMAD2‐S467 (AP0269; ABclonal), SMAD2 (A19114; ABclonal), phospho‐SMAD3‐S423/S425 (AP1263; ABclonal), SMAD3 (A19115; ABclonal), phospho‐SMAD1/5/8 (#13 820; Cell Signaling Technology), SMAD15/8 (A17439; ABclonal), BMPR2 (sc‐393304; Santa Cruz Biotechnology), ALK3 (sc‐134285; Santa Cruz Biotechnology), SMAD7 (sc‐365846; Santa Cruz Biotechnology).

Techniques: Expressing, Binding Assay, Incubation, Transfection, Western Blot, Immunoprecipitation, Spectroscopy

Journal: Advanced Science

Article Title: Autocrine GDF10 Inhibits Hepatic Stellate Cell Activation via BMPR2/ALK3 Receptor to Prevent Liver Fibrosis

doi: 10.1002/advs.202500616

Figure Lengend Snippet: GDF10 inhibits HSC activation via the BMPR2/ALK3‐SMAD1/5/8‐SMAD7 signaling pathway. A,B) qPCR (A) ( n = 6) and Western blot (B) analysis of indicated genes in the primary mouse HSCs infected with LV‐sh‐Luc or LV‐sh‐Bmpr2 and LV‐sh‐Alk3 for 24 h and then treated with TGF‐β1 plus Fc or GDF10‐Fc for another 24 h. C) IF staining analysis of ACTA2 expression in HSCs treated as in (A), scale bars, 20 µm ( n = 3). D, E) qPCR ( n = 6) (D) and Western blot (E) analysis of indicated genes in the HSCs treated with TGF‐β1 plus GDF10‐Fc and/or LDN for 24 h. F) IF analysis of ACTA2 stating in HSCs treated as in (D). G) Heat map shows the indicated genes in JS1 cells treated with TGF‐β1 plus LDN and/or GDF10‐Fc for 24 h. H) qPCR (top) ( n = 6) and Western blot (bottom) analysis of SMAD7 mRNA and protein levels in HSCs treated as in (G). I) qPCR (top) ( n = 6) and Western blot (bottom) analysis of SMAD7 mRNA and protein levels in HSCs treated with TGF‐β1 plus DM and/or GDF10‐Fc for 24 h. J) Alignment of the SMAD7 promoter regions containing the GC‐BRE sites (GGCGCC) in the indicated species. K) Luciferase reporter gene assay in JS1 cells transfected with the indicated plasmids and then treated with Fc or GDF10‐Fc. L) ChIP assay showing the recruitment of phosphorylated SMAD1/5/8 to the Smad7 gene promoter in JS1 cells ( n = 3). M,N) qPCR (M) ( n = 6) and Western blot (N) analysis of indicated genes in the primary mouse HSCs infected with LV‐sh‐Luc or LV‐sh‐Smad7 for 24 h, then treated with TGF‐β1 plus Fc or GDF10‐Fc for another 24 h. O) IF staining analysis of ACTA2 expression in HSCs treated as in (M), scale bars, 20 µm ( n = 3). Data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 by the two‐tailed Student's t ‐test (K,L), one‐way ANOVA (C,F,H,I,O), or two‐way ANOVA (A,D,M).

Article Snippet: For Western blot analysis, the following primary antibodies were employed: β‐Actin (AC026; ABclonal), GDF10 (ab235005; Abcam), ACTA2 (A17910; ABclonal), COL1A1 (A22089; ABclonal), phospho‐SMAD2‐S467 (AP0269; ABclonal), SMAD2 (A19114; ABclonal), phospho‐SMAD3‐S423/S425 (AP1263; ABclonal), SMAD3 (A19115; ABclonal), phospho‐SMAD1/5/8 (#13 820; Cell Signaling Technology), SMAD15/8 (A17439; ABclonal), BMPR2 (sc‐393304; Santa Cruz Biotechnology), ALK3 (sc‐134285; Santa Cruz Biotechnology), SMAD7 (sc‐365846; Santa Cruz Biotechnology).

Techniques: Activation Assay, Western Blot, Infection, Staining, Expressing, Luciferase, Reporter Gene Assay, Transfection, Two Tailed Test

Journal: Advanced Science

Article Title: Autocrine GDF10 Inhibits Hepatic Stellate Cell Activation via BMPR2/ALK3 Receptor to Prevent Liver Fibrosis

doi: 10.1002/advs.202500616

Figure Lengend Snippet: BMPR2:ALK3‐Fc prevents the antifibrotic effect of GDF10 in the liver. A) Schematic drawing of the experimental procedure in 3‐month‐old male LoxP and Gdf10TG mice. B) Representative images of H&E, Sirius Red, and ACTA2 IHC staining, scale bars, 50 µm ( n = 3). C) Hydroxyproline assay analysis of the total liver collagen of mice treated as in (A) ( n = 6). D,E) qPCR (D) ( n = 6) and Western blot (E) analysis of indicated genes in the liver of mice treated as in (A). F,G) qPCR (F) ( n = 6) and Western blot (G) analysis of indicated genes in the HSCs from mice treated as in (A) ( n = 3). H,I) IF staining (H) and Western blot (I) analysis of indicated protein in the HSCs from mice treated as in (A), scale bars, 5 µm. Data are mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 by the one‐way ANOVA (C,E,G,I) or two‐way ANOVA (D,F).

Article Snippet: For Western blot analysis, the following primary antibodies were employed: β‐Actin (AC026; ABclonal), GDF10 (ab235005; Abcam), ACTA2 (A17910; ABclonal), COL1A1 (A22089; ABclonal), phospho‐SMAD2‐S467 (AP0269; ABclonal), SMAD2 (A19114; ABclonal), phospho‐SMAD3‐S423/S425 (AP1263; ABclonal), SMAD3 (A19115; ABclonal), phospho‐SMAD1/5/8 (#13 820; Cell Signaling Technology), SMAD15/8 (A17439; ABclonal), BMPR2 (sc‐393304; Santa Cruz Biotechnology), ALK3 (sc‐134285; Santa Cruz Biotechnology), SMAD7 (sc‐365846; Santa Cruz Biotechnology).

Techniques: Immunohistochemistry, Hydroxyproline Assay, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Hepcidin expression is associated with increased γ-secretase–mediated cleavage of neogenin in the liver

doi: 10.1016/j.jbc.2024.107927

Figure Lengend Snippet: Hjv prevents Netrin-1–induced Neo1 degradation, and Alk3 facilitates the generation of γ-secretase–cleaved Neo1-ECD/TMD in Hep3B cells. A , diagram of fNeo1, mouse Hjv with an N-terminal 3xFLAG epitope (fHjv), mouse Alk3 with an N-terminal FLAG/MYC epitope (fAlk3), and mouse Netrin-1. B , co-expression with fHjv does not inhibit α-secretase cleavage of fNeo1. At 56 h after transfection, cell surface proteins were biotinylated. The eluted cell surface proteins, ∼10% of input lysate, and a fraction of concentrated CM were subjected to SDS-PAGE and immunodetection (IB). C , co-expression with fAlk3 increases fNeo1, fNeo1-ECD/TMD, and fNeo1-ECD. Experiments were performed as described above in ( B ) except that LY450139 (10 μM) was used to inhibit γ-secretase proteolysis. Each panel was cropped from the same image. D , quantification of cell surface fNeo1 and fNeo1-ECD/TMD bands in ( C ) (panel-1, lane 7/9), as well as fNeo1-ECD bands in ( C ) (lowest panel, lane 2/4) (n = 3). E , co-expression with fAlk3 increases γ-secretase–cleaved fNeo1-ICD. Experiments were performed as described in the legend to Fig. 3 B . Each panel was cropped from the same image. Two sets of images with different exposure for anti-FLAG antibody were presented. F , quantification of fNeo1-ICD bands in ( E ) (panel-2; lane 5/6) (n = 3). G , incubation with Netrin-1 decreases fNeo1 levels in Hep3B cells. fNeo1-transfected Hep3B cells were incubated with mouse Netrin-1 at 0, 0.1, 0.25, 0.5, or 1.0 μg/ml for ∼16 h. Cell surface proteins were subjected to biotinylation and immunodetection. H , quantification of total cell surface fNeo1 in ( G ) (panel-4) (n = 3). I , co-expression of Hjv and Neo1 prevents Netrin-1–mediated degradation of Neo1. Cells were transfected as described above in ( B ) and incubated with mouse Netrin-1 at 0 and 0.5 μg/ml for ∼16 h, followed by biotinylation and immunodetection. All experiments were repeated at least three times with consistent results. All quantification data shown are means ± SD. ∗, p < 0.05; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Article Snippet: Mouse Neo1 ORF (NM_008684) with a C-terminal FLAG/MYC epitope (fNeo1) in pCMV6 vector (#MR226235) and mouse Alk3 ORF (NM_009758.3) with a C-terminal FLAG/MYC epitope (fAlk3) in pCMV6 vector (#MR227586) were obtained from OriGene Technologies Inc. We obtained pEGFP-N1 plasmid from Clontech.

Techniques: Expressing, Transfection, SDS Page, Immunodetection, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Hepcidin expression is associated with increased γ-secretase–mediated cleavage of neogenin in the liver

doi: 10.1016/j.jbc.2024.107927

Figure Lengend Snippet: Deletion of the Neo1 Ig-like domains compromises its ability to induce hepcidin expression. A , diagrams of mouse fNeo1 and fNeo1 ΔIg constructs. B , qRT-PCR analysis of hepatic Neo1 mRNA from PBS-injected Neo1 fl/fl ;Alb-Cre − male mice, PBS-injected Neo1 fl/fl ;Alb-Cre + male mice (−), and Neo1 fl/fl ;Alb-Cre + male mice transduced with AAV8-fNeo1 and male fNeo1 ΔIg . C , representative images of Western blot analysis for transduced fNeo1, fNeo1 ΔIg , and endogenous Tfr2 from the mice as described above in ( B ). Images in panels-1/2 were derived from the immunodetection of liver membrane extracts (250 μg protein). The image in panel-3 was obtained from the same membrane extracts, except that ∼2 mg extract proteins from each sample were first subjected to pull down by using anti-FLAG affinity gel, followed by immunodetection with an anti-FLAG antibody. D - E , qRT-PCR analysis of hepatic hepcidin and Id1 mRNA. F , serum iron (Fe) assay. G , liver nonheme iron (Fe) assay. Each group consists of at least seven animals. All qRT-PCR results are expressed as the amount relative to that of β-actin for each sample. Data shown are means ± SD. One-way ANOVA and Tukey’s post-test were used to analyze the data relative to PBS-injected Neo1 fl/fl ;Alb-Cre - mice. In addition, the analyzed results between fNeo1 and fNeo1 ΔIg groups were also presented. ∗, p < 0.05; ∗∗ p < 0.01; ∗∗∗, p < 0.001. H - J , models for the induction of hepcidin expression by hepatocyte Neo1 via its interaction with Hjv, Alk3, and other hepcidin-inducing proteins. PM, plasma membrane; n.s., nonspecific band.

Article Snippet: Mouse Neo1 ORF (NM_008684) with a C-terminal FLAG/MYC epitope (fNeo1) in pCMV6 vector (#MR226235) and mouse Alk3 ORF (NM_009758.3) with a C-terminal FLAG/MYC epitope (fAlk3) in pCMV6 vector (#MR227586) were obtained from OriGene Technologies Inc. We obtained pEGFP-N1 plasmid from Clontech.

Techniques: Expressing, Construct, Quantitative RT-PCR, Injection, Transduction, Western Blot, Derivative Assay, Immunodetection, Membrane