Journal: Micromachines

Article Title: A Nitrocellulose Paper-Based Multi-Well Plate for Point-of-Care ELISA

doi: 10.3390/mi13122232

Figure Lengend Snippet: The schematic representation of the ( a ) direct ELISA of rabbit IgG and ( b ) the sandwich ELISA of sperm protein SP-10 on the NC paper multi-well plate.

Article Snippet: The sandwich ELISA protocol is described as follows: The sandwich ELISA of SP-10 starts with the addition of 3 μL of the capture antibody solution (mouse anti-ACRV1 monoclonal antibody in CBS solution, cat #11789-MM01, Sino Biological) with a concentration of 100 μg/mL, onto the NC paper well, followed by the overnight incubation in a 4 °C fridge for the sufficient antibody immobilization, Then, after taking the blocked plate out of the fridge, each NC paper well was pipetted with 10 μL of the wash buffer (0.05% Tween20 in TBS, pH 7.2–7.4) and blotted with an absorbent pad, three times, to remove the unbound antibodies, Next, 3 μL of the blocking buffer (2% BSA in wash buffer) was added to each well ( (b-i)), followed by a 10-min incubation and washing step with 10 μL wash buffer, three times, again to remove the unbound blocking reagents ( (b-ii)), Next, 3 μL of the SP-10 protein solution (cat #10227-H08B, Sino Biological) was added to the blocked NC paper well and incubated for 10 min, for the SP-10 protein to be captured by the immobilized capture antibody probe ( (b-iii)).

Techniques: Direct ELISA, Sandwich ELISA

Journal: REPRODUCTION

Article Title: Postnatal uterine development in Inverdale ewe lambs

doi: 10.1530/rep-07-0323

Figure Lengend Snippet: Figure 3 Immunohistochemical localization of androgen receptor (AR) and activin receptors (ActRIA, IB and II) in the uteri of CC and II ewes. GE, glandular epithelium; LE, luminal epithelium; S, stroma. The scale bar indicates 100 mm at low magnification and 25 mm at high magnification.

Article Snippet: Rabbit polyclonal antibody to androgen receptor (sc-816, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and mouse monoclonal Reproduction (2008) 135 357–365 antibodies to ActRIA (MAB637; R&D Systems Inc.), ActRIB (MAB222; R&D Systems), and ActRII (MAB3391; R&D Systems) were used for immunohistochemistry (Carpenter et al. 2003a, Hayashi et al. 2003, Juengel et al. 2006).

Techniques: Immunohistochemical staining

Journal: The Journal of Clinical Investigation

Article Title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro-adipogenic progenitors in fibrodysplasia ossificans progressiva mice

doi: 10.1172/JCI153795

Figure Lengend Snippet: ( A ) Mouse C2C12 myoblast cells express similar levels of Acvr1 and Alk3 , low levels of Alk1 , and no detectable Alk6 , as quantified by RT-qPCR ( n = 3). ΔCT values were calculated using the average CT values of the internal controls, Gapdh or Actb (β-actin) (see Methods). Error bars represent ±SD. CT values >40 were considered not detected (N.D.). ( B ) Surface expression of ACVR1 and ALK3 on C2C12 cells, as detected by flow cytometry. ( C ) mAb JAB0505 binds to parental C2C12 cells, but not Acvr1 -KO cells, as assessed by flow cytometry. ( D ) JAB0505 inhibits BMP9-induced signal activation in wild-type and ACVR1(R206H)-overexpressing C2C12 cells in a dose-dependent manner, as determined by quantification of BRE-luciferase activity ( n = 3). Error bars represent ±SD.

Article Snippet: Cells were removed from adherent cultures using AssayComplete cell dissociation reagent (Eurofins, 92-0009) and incubated with a polyclonal anti-ALK2 antibody (R&D Systems, AF637) or a polyclonal anti-ALK3 antibody (Sino Biological, 50078-RP02).

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Activation Assay, Luciferase, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro-adipogenic progenitors in fibrodysplasia ossificans progressiva mice

doi: 10.1172/JCI153795

Figure Lengend Snippet: ( A ) Representative μCT images of HO in Acvr1 FLEx(R206H)/+ ; CAG-Cre ERT2 mice 20 days after cardiotoxin-induced injury of the gastrocnemius muscle (Untreated, n = 3; JAB0505, n = 4). ( B ) Representative μCT images of HO (pseudocolored green) in Acvr1 tnR206H/+ ; Tie2-Cre mice 21 days after pinch injury of the gastrocnemius muscle (Untreated, n = 11; JAB0505, n = 10). ( C ) Quantification of HO volumes as a function of time after muscle pinch injury of Acvr1 tnR206H/+ ; Tie2-Cre mice. Untreated, n = 11; JAB0505 (10 mg/kg), n = 6. Error bars represent ±SEM. **** P ≤ 0.0001 by 2-way ANOVA with Sidak’s multiple-comparison test. ( D ) Paired single transverse slice and 3D reconstructed μCT images of the distal hind limb of Acvr1 tnR206H/+ ; Tie2-Cre mice at the indicated times after hind limb muscle pinch injury with and without administration of JAB0505. Mineralized bone in the slice images is pseudocolored green. Radio-opaque lesional tissue below the threshold set for quantification of mineralized bone (white arrows in day 14 slices) is extensive at day 14 in JAB0505-treated mice. Mineralized bone in day 14 slices is barely visible at this magnification. HO volumes are given for images prior to day 35. The tibia and fibula are labeled with asterisks in the day 14 slices. Pelvic bones present in day 21, 28, and 35 slices of JAB0505-treated mice are denoted with arrowheads. To avoid confusion with HO, the baculum present in some images was removed by segmentation.

Article Snippet: Cells were removed from adherent cultures using AssayComplete cell dissociation reagent (Eurofins, 92-0009) and incubated with a polyclonal anti-ALK2 antibody (R&D Systems, AF637) or a polyclonal anti-ALK3 antibody (Sino Biological, 50078-RP02).

Techniques: Comparison, Labeling

Journal: The Journal of Clinical Investigation

Article Title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro-adipogenic progenitors in fibrodysplasia ossificans progressiva mice

doi: 10.1172/JCI153795

Figure Lengend Snippet: ( A ) Transverse sections of muscle from untreated and JAB0505-treated Acvr1 tnR206H/+ ; Tie2-Cre mice on days 6 and 14 after muscle pinch injury. Alcian blue staining to detect cartilage (blue) and immunohistochemical staining to detect ACVR1 (brown) were performed on nearby sections. Sections processed for Alcian blue were counterstained with eosin, and sections processed for ACVR1 immunohistochemistry were counterstained with hematoxylin. On day 6 after injury, untreated Acvr1 tnR206H/+ ; Tie2-Cre mice exhibited a spatially discrete lesional region (asterisk) that was primarily comprised of ACVR1-positive ectopic cartilage. By day 14, the lesional region (asterisk) of untreated Acvr1 tnR206H/+ ; Tie2-Cre mice displayed sporadic ACVR1 localization and was composed of both cartilage and morphologically apparent bone. In contrast, JAB0505 treated Acvr1 tnR206H/+ ; Tie2-Cre mice displayed multiple apparent cartilaginous lesions and broader distribution of ACVR1 localization on days 6 and 14 (arrows). Centrally located myofiber nuclei (arrowheads), which identify regenerated fibers, were rare in Acvr1 tnR206H/+ ; Tie2-Cre mice, and undetected in JAB0505-treated Acvr1 tnR206H/+ ; Tie2-Cre mice. AB/E, Alcian blue/eosin. Original magnification, ×100. ( B ) Transverse sections of lower hind limbs of Acvr1 tnR206H/+ ; Tie2-Cre mice 14 days after injury. Alcian blue staining revealed numerous cartilaginous lesions (blue, examples at arrows) in injured muscle of JAB0505-treated mice. Sections were counterstained with eosin. T, tibia. Original magnification, ×40.

Article Snippet: Cells were removed from adherent cultures using AssayComplete cell dissociation reagent (Eurofins, 92-0009) and incubated with a polyclonal anti-ALK2 antibody (R&D Systems, AF637) or a polyclonal anti-ALK3 antibody (Sino Biological, 50078-RP02).

Techniques: Staining, Immunohistochemical staining, Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro-adipogenic progenitors in fibrodysplasia ossificans progressiva mice

doi: 10.1172/JCI153795

Figure Lengend Snippet: μCT images of the distal hind limbs of 4 Acvr1 tnR206H/+ ; Tie2-Cre FOP mice (numbered 1–4) at the indicated time points after injection of 50 μL of 2.5% methylcellulose into the tibialis anterior muscle, with and without administration of 10 mg/kg JAB0505 ( n = 2 mice, 4 injected limbs, for each group). HO is pseudocolored green. A lateral view of the right hind limb of each mouse is shown. Contralateral hind limbs (not shown) received equivalent injuries and the extent of HO was comparable.

Article Snippet: Cells were removed from adherent cultures using AssayComplete cell dissociation reagent (Eurofins, 92-0009) and incubated with a polyclonal anti-ALK2 antibody (R&D Systems, AF637) or a polyclonal anti-ALK3 antibody (Sino Biological, 50078-RP02).

Techniques: Injection

Journal: The Journal of Clinical Investigation

Article Title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro-adipogenic progenitors in fibrodysplasia ossificans progressiva mice

doi: 10.1172/JCI153795

Figure Lengend Snippet: ( A ) Osteogenic differentiation of monolayer R206H-FAP cultures, as assessed by ALP staining (purple), and ( B ) chondrogenic differentiation of micromass cultures assessed by Alcian blue staining. ActA-mAb was used at 1 μg/mL (7 nM) and JAB0505 was used at 10 μg/mL (~70 nM). ( C ) Western blot of phosphorylated SMAD1/5/8 (p-SMAD1/5/8) in wild-type (WT) and R206H-FAPs (R206H). β-Actin was used as a loading control. ( D ) μCT of the distal hind limb of Acvr1 tnR206H/+ ; Tie2-Cre mice on day 21 after injury. At the time of muscle injury, mice were treated with ActA-mAb (10 mg/kg) alone or ActA-mAb with JAB0505 (10 mg/kg). HO is pseudocolored green, and quantification is shown. ActA-mAb, n = 6; JAB0505 plus ActA-mAb, n = 6. Error bars represent ±SD. **** P < 0.0001 by 2-tailed, unpaired t test. ( E ) μCT images of the distal hind limb 21 days after transplantation of R206H-FAPs into the injured gastrocnemius of SCID hosts. ActA-mAb (10 mg/kg) and JAB0505 (10 mg/kg) were administered at the time of transplantation. HO is pseudocolored green and quantified, with error bars representing ±SD. Untreated, n = 10; JAB0505, n = 16; ActA-mAb, n = 6; JAB0505 plus ActA-mAb, n = 8.

Article Snippet: Cells were removed from adherent cultures using AssayComplete cell dissociation reagent (Eurofins, 92-0009) and incubated with a polyclonal anti-ALK2 antibody (R&D Systems, AF637) or a polyclonal anti-ALK3 antibody (Sino Biological, 50078-RP02).

Techniques: Staining, Western Blot, Control, Transplantation Assay

Journal: The Journal of Clinical Investigation

Article Title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro-adipogenic progenitors in fibrodysplasia ossificans progressiva mice

doi: 10.1172/JCI153795

Figure Lengend Snippet: ( A ) 3D tomographic bioluminescence source reconstruction following muscle pinch injury of Acvr1 tnR206H/+ ; R26 Luc/+ ; Tie2-Cre FOP mice, with and without administration of JAB0505. Paired images show μCT alone (left panel) and μCT combined with the corresponding 3D bioluminescence reconstruction (right panel). The same mouse is shown from days 3 to 21. Bioluminescence reconstruction was not performed on day 21 due to the dampening effect of dense bone on luminescent output. ( B ) Graphical representation of bioluminescent population dynamics of Tie2 + cells from Acvr1 tnR206H/+ ; R26 Luc/+ ; Tie2-Cre mice following pinch injury. Untreated, n = 16; JAB0505, n = 10. Error bars represent ±SEM. *** P ≤ 0.001, **** P ≤ 0.0001 by 2-way ANOVA with Sidak’s multiple-comparison test. ( C ) Flow cytometry analysis to determine R206H-FAP cell number in injured distal hind limb muscles of Acvr1 tnR206H/+ ; R26 NG/+ ; Tie2-Cre mice that were either untreated (day 5, n = 4; day 10, n = 9) or administered JAB0505 at 10 mg/kg (day 5, n = 4; day 10, n = 10). Error bars represent ±SD. ** P ≤ 0.01 by 2-tailed, unpaired t test with Welch’s correction.

Article Snippet: Cells were removed from adherent cultures using AssayComplete cell dissociation reagent (Eurofins, 92-0009) and incubated with a polyclonal anti-ALK2 antibody (R&D Systems, AF637) or a polyclonal anti-ALK3 antibody (Sino Biological, 50078-RP02).

Techniques: Comparison, Flow Cytometry, Muscles

Journal: The Journal of Clinical Investigation

Article Title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro-adipogenic progenitors in fibrodysplasia ossificans progressiva mice

doi: 10.1172/JCI153795

Figure Lengend Snippet: Flow cytometry analysis was used to determine cell numbers of ( A ) total CD45 + hematopoietic cells, ( B ) myeloid cells, ( C ) lymphoid cells, ( D ) total macrophages, ( E ) Ly6C + inflammatory monocytes/macrophages, ( F ) neutrophils, ( G ) mast cells, and ( H ) T cells in injured distal hind limb muscles of control ( R26 NG/+ ; Tie2-Cre) and FOP ( Acvr1 tnR206H/+ ; R26 NG/+ ; Tie2-Cre) mice that were either untreated or administered 10 mg/kg JAB0505 i.p. ( n = 3–4). Error bars represent ±SD. Significance was determined using 1-way ANOVA with Tukey’s multiple-comparison test within individual time points. Symbols representing significance were placed above FOP and FOP + JAB0505 bars to indicate a comparison to control (^), control + JAB0505 ( # ), and FOP (*). The numbers of symbols of each type denote levels of significance: P ≤ 0.05, P ≤ 0.01, P ≤ 0.001, and P ≤ 0.0001. No control vs. control + JAB0505 comparisons were significant.

Article Snippet: Cells were removed from adherent cultures using AssayComplete cell dissociation reagent (Eurofins, 92-0009) and incubated with a polyclonal anti-ALK2 antibody (R&D Systems, AF637) or a polyclonal anti-ALK3 antibody (Sino Biological, 50078-RP02).

Techniques: Flow Cytometry, Muscles, Control, Comparison

Journal: Biology of reproduction

Article Title: The activin-follistatin system in the neonatal ovine uterus.

doi: 10.1095/biolreprod.103.016287

Figure Lengend Snippet: FIG. 2. Expression of ActRs in the neonatal ovine uterus. In situ hybridization and immunohistochemical analysis of ActRIA (A) and ActRII (B) and immunohistochemical analysis of ActRIB (C) in the uterus. Except for ActRIB, representative photomicrographs of in situ hybridization results are presented in bright-field and dark-field illumination (left). Melanocytes (Mel) in the endometrium appear white in the dark-field images and black in bright-field images, but they do not express subunit mRNA. Representative photomicrographs of immunohistochemical results are presented for the upper and lower portions of the uterine wall (right). As a negative control, mouse IgG (mIgG) was substituted for the primary antibody. M, Myometrium; S, stroma. Bars 5 50 mm.

Article Snippet: Mouse anti-human monoclonal antibody to follistatin (catalog no. MAB669), ActRIA (catalog no. MAB637), ActRIB (catalog no. MAB222), and ActRIIA/B (catalog no. MAB3391) were from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Expressing, In Situ Hybridization, Immunohistochemical staining, Negative Control

Journal: American Journal of Hematology

Article Title: A Recombinant Antibody Against ALK2 Promotes Tissue Iron Redistribution and Contributes to Anemia Resolution in a Mouse Model of Anemia of Inflammation

doi: 10.1002/ajh.27578

Figure Lengend Snippet: RKER‐216 epitope in ALK2 overlaps with BMP6 binding site, and the presence of RKER‐216 competitively inhibits BMP6 from binding to ALK2. (A, E) Binding affinity ( K D ) between ALK2 and RKER‐216 or ALK2 and BMP6 was determined by SPR. RKER‐216 diluted in HBS‐EP+ buffer was injected through a CM4 chip immobilized with ALK2‐Fc at 25 RU (A) while BMP6 diluted in HBS‐EP+ buffer with 30 nM arginine was injected through a CM4 chip immobilized with ALK2‐Fc at 350 RU (E). The results were analyzed by Biacore Insight Evaluation software using 1:1 binding model for RKER‐216 and steady‐state affinity for BMP6. The mean K D ± SEM from 3 separate experiments is reported and a representative sensogram is shown. (B) RKER‐216 binds to regions in ALK2 covering the F2 loop and β4 sheet (blue) as determined by HDX‐MS. The H/D exchange protected regions (blue) were mapped onto a model structure of ALK2 (PDB ID: 7YRU) shown in ribbon (left) and in surface (right) representation in complex with BMP6 structure (PBD ID: 2R52). The model of ALK2:BMP6 complex was generated by aligning individual structure of ALK2 and BMP6 into the structure of ALK1:BMP9 (PDB ID: 4FAO). The sequence alignment between ALK2 and ALK3 are shown (bottom), highlighting the binding region (in blue) of RKER‐216 in ALK2. (C‐D) BLI competition assays were performed in two ways. (C) ALK2 binding site was saturated with 5 nM of RKER‐216 prior to the competition with BMP6 at 0 to 100 nM. In addition to the vehicle control, BMP6 at 100 nM was added to unsaturated ALK2 to confirm binding ability of BMP6 in the competition. No binding activity was observed in the competition, indicating that BMP6 at 100 nM was unable to complete RKER‐216 away for binding ALK2. (D) ALK2 binding site was saturated with 100 nM of BMP6 prior to the competition with RKER‐216 at 0 to 50 nM. RKER‐216 at 50 nM was added to unsaturated ALK2 to confirm binding ability of RKER‐216. This group also served as a control for examining whether RKER‐216 binds to ALK2 or the ALK2‐BMP6 complex, as changes in the wavelength reflects both the size and affinity of the interactor. In the zoomed plot of RKER‐216 competition, data were aligned to zero on the Y ‐axis prior to the competition step for visual comparison, and 50 nM RKER‐216 without saturation had the greatest wavelength shift, indicating that RKER‐216 dislodged BMP6 from ALK2 instead of binding to the ALK2‐BMP6 complex. Experiments were repeated 3 times and a representative sensogram is shown.

Article Snippet: Briefly, the library was panned for antibodies recognizing the extracellular domain of human ALK2 (637‐AR; R&D Systems) with three rounds of enrichment and counter‐selected against the Fc control for depletion.

Techniques: Binding Assay, Injection, Software, Generated, Sequencing, Control, Activity Assay, Comparison

Journal: American Journal of Hematology

Article Title: A Recombinant Antibody Against ALK2 Promotes Tissue Iron Redistribution and Contributes to Anemia Resolution in a Mouse Model of Anemia of Inflammation

doi: 10.1002/ajh.27578

Figure Lengend Snippet: RKER‐216 decreases hepcidin transcription in vitro and controls iron availability by lowering hepcidin secretion in vivo. (A) Hep3B cells were serum starved with 1% FBS overnight and incubated with an ascending dose of RKER‐216 (0.02–1 μg/mL) in the absence or presence of 5 ng/mL of BMP6, BMP2/6, or BMP2 for 6 h ( n = 4–5 per group). (B) Characterization of human ACVR1 knockout in HepG2 and Huh7 cells by quantifying ACVR1 and BMPR1A transcript copy number ( n = 3 per group). (C) ACVR1 KO and the negative control cells were treated with RKER‐216 at 1–30 μg/mL for 6 h ( n = 3 per group). Relative HAMP mRNA levels were determined by qRT‐PCR and transcripts were normalized to an internal control RPL19 . The average of PBS control without ligand stimulation or ACVR1 wildtype without RKER‐216 was set to 1. For ligand preference, hepcidin stimulated by each ligand was set to 100%. Values represent mean ± SEM. Results were compared across RKER‐216 and BMP ligands by one‐ or two‐way ANOVA with Tukey's post hoc test. Means without a common superscript differ significantly ( p < 0.05) or * p < 0.05, *** p < 0.001 relative to untreated cells of the same genotype. (D‐G) B6N male mice at 8 weeks were treated with a single SC dose of isotype control or RKER‐216 at 3 mg/kg for times as indicated ( n = 5 per group). Serum was collected to quantify (D) RKER‐216 exposure and (E) serum hepcidin by ELISA, and (F) serum iron and (G) serum transferrin saturation (TSAT) by colorimetric assays. Values represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 relative to the isotype control mice of the same time point by Student's t ‐test.

Article Snippet: Briefly, the library was panned for antibodies recognizing the extracellular domain of human ALK2 (637‐AR; R&D Systems) with three rounds of enrichment and counter‐selected against the Fc control for depletion.

Techniques: In Vitro, In Vivo, Incubation, Knock-Out, Negative Control, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ALK2-WT, but not ALK2-R206H, forms a significant amount of homomeric complexes. COS7 cells were co-transfected with expression vectors encoding myc-tagged ALK2-WT or ALK2-R206H alone or together with their HA-tagged counterpart (or empty vector). Where shown, the HA-tagged receptor was immobilized by IgG-crosslinking as in , following the schematic description in . Thus, the HA-tagged receptor was patched and crosslinked by IgGs, and the myc-tagged version of the same receptor was labeled by Fab’ fragments. The lateral mobility of the Fab’-labeled myc-tagged receptor was measured by FRAP. Where indicated, ActA (4 nM) was added where indicated as in . ( A , C ) Average R f values; ( B , D ) average D values. Bars are mean ± SEM; the number of measurements (each conducted on a different cell) is shown within each bar. Asterisks indicate significant differences between the R f values of the pairs indicated by brackets (*, p < 0.03; one-way ANOVA and Bonferroni post hoc test. n.s. = not significant). A similar analysis of the D values showed no significant differences.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Labeling

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2B forms stable heteromeric complexes with either ALK2-WT or ALK2-R206H. COS7 cells were co-transfected with expression vectors encoding myc-ACVR2B alone or together with HA-tagged ALK2 (WT or R206H). Where indicated, HA-ALK2 was immobilized by IgG-crosslinking as in , as shown schematically in . The HA-tagged receptor (ALK2-WT or ALK2-R206H) was patched and crosslinked by IgGs, and the co-expressed myc-ACVR2B was labeled exclusively by monovalent Fab’; the lateral mobility of Fab’-labeled myc-ACVR2B was measured by FRAP, without or with ActA (4 nM). ( A ) Quantification of the cell surface levels of myc-ACVR2B alone or co-expressed with HA-tagged ALK2 variants. Myc-ACVR2B cell surface receptors were labeled at 4 °C by a saturating concentration (40 μg/mL) of murine Fab’ αmyc, followed by 40 μg/mL Alexa 546-Fab’ GαM, and fixed (4% paraformaldehyde). This protocol enables the measurement of the levels of the tagged receptors at the plasma membrane under identical conditions (same laser excitation line and intensity, same microscope filters, same settings of the photomultiplier tube) [ , ]. The surface levels of the receptors were quantified by measuring the fluorescence intensity from a point-confocal spot by the FRAP apparatus under non-bleaching conditions as described) [ , ]. Data are mean ± SEM of 30 measurements under each condition. No significant differences were observed between the values under the different conditions. ( B , D ) Average R f values; ( C , E ) average D values. Bars depict the average values (mean ± SEM); the number of measurements (each conducted on a different cell) is shown on each bar. Asterisks indicate significant differences between the R f values of the pairs indicated by brackets (**, p < 4 × 10 −3 ; ***, p < 7 × 10 −4 ; ****, p < 10 −4 ; one-way ANOVA and Bonferroni post hoc test. n.s. = not significant). Similar analysis of the D values showed no significant differences.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Transfection, Expressing, Labeling, Concentration Assay, Membrane, Microscopy, Fluorescence

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2A forms heteromeric complexes with ALK2-WT to a much higher degree than with ALK2-R206H. Experiments were as in , following the scheme depicted in , except that Fab’-labeled myc-ACVR2A replaced myc-ACVR2B. Where indicated, HA-ALK2-WT or HA-ALK2-R206H were immobilized by IgG-crosslinking, as in . The lateral mobility of Fab’-labeled myc-ACVR2A was measured by FRAP, without or with ActA (4 nM; see ). ( A ) Quantification of the cell surface levels of myc-ACVR2A alone or co-expressed with HA-tagged ALK2 variants. The experiment was conducted exactly as described in A. Data are mean ± SEM of 30 measurements under each condition. No significant differences were observed between the surface levels of myc-ACVR2A alone or co-expressed with HA-tagged ALK2 (WT or R206H). ( B , D ) Average R f values; ( C , E ) average D values. Bars depict the average values (mean ± SEM); the number of measurements (each conducted on a different cell) is shown on each bar. Asterisks indicate significant differences between the R f values of the pairs indicated by brackets (*, p < 0.03; ***, p < 8 × 10 −4 ; ****, p < 10 −4 ; one-way ANOVA and Bonferroni post hoc test. n.s. = not significant). Similar analysis of the D values showed no significant differences.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Labeling

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2B, but not ACVR2A, enhances ALK2-WT homomeric interactions in the presence of ActA. Patch/FRAP studies were conducted on COS7 cells expressing myc-ALK2-WT alone or together with HA-ALK2-WT. Where indicated, untagged ACVR2A, ACVR2B, or ACVR2B-KD were co-expressed. CL marks IgG-mediated crosslinking of HA-ALK2-WT, performed as in A,B. The schematics of the experimental design are depicted in ; HA-ALK2-WT was patched and crosslinked by IgGs, while myc-ALK2-WT was labeled by Fab’ fragments. Where indicated, an untagged ACVR2 receptor variant was co-expressed as a third receptor to measure its effect. The lateral mobility of the Fab’-labeled myc-ALK2-WT was measured by FRAP. ( A ) Control experiments showing that the cell surface levels of myc-ALK2-WT are not significantly affected by co-expression with untagged ACVR2A or ACVR2B. The experiment was conducted as described in A, except that the surface levels of myc-ALK2-WT were measured by the point confocal method (see A). Data are mean ± SEM of 30 measurements in each case. No significant differences were observed between the surface levels of myc-ALK2-WT alone or co-expressed with untagged ACVR2A or ACVR2B (one-way ANOVA and Bonferroni post hoc test). ( B ) Average R f values; ( C ) average D values. Bars depict the average values (mean ± SEM); the number of measurements is depicted within each bar. Asterisks indicate significant differences between the R f values of the pairs indicated by brackets (*, p < 0.03; **, p < 9 × 10 −3 ; ***, p < 2 × 10 −4 ; one-way ANOVA and Bonferroni post hoc test. n.s. = not significant). Similar analysis of the D values showed no significant differences. The left panels in ( B , C ), designated “-Untag. Type II”, depict the R f and D values of myc-ALK2-WT co-expressed with HA-ALK2-WT without or with IgG αHA crosslinking; these values were taken from A,B and are shown to enable direct comparison with the effects of co-expressing untagged ACVR2A/B. As shown, co-expression with untagged ACVR2A interfered with the formation of homomeric ALK2-WT complexes, which were restored in the presence of ActA (which dimerizes ACVR2A; G). On the other hand, ACVR2B (which forms homodimers also without ActA; E) did not disrupt ALK2-WT homomeric interactions and elevated them in the presence of ActA. Of note, ACVR2B-KD had the same effects as ACVR2B, demonstrating that the kinase activity of the type II receptor is not required.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Expressing, Labeling, Variant Assay, Comparison, Activity Assay

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2B is more effective than ACVR2A in mediating ALK2-R206H homomeric interactions. Patch/FRAP studies were conducted exactly as described in , except that myc-and HA-tagged ALK2-R206H replaced the tagged ALK2-WT constructs. The experimental scheme follows the one depicted in , as explained in . The lateral mobility of the Fab’-labeled myc-ALK2-R206H was measured by FRAP. ( A ) The cell surface levels of myc-ALK2-R206H are not altered by co-expression with untagged ACVR2A or ACVR2B. The experiment was conducted as described in A, except that the surface levels of myc-ALK2-R206H were measured by the point confocal method (see A). Data are mean ± SEM of 30 measurements in each case. No significant differences were observed between the surface levels of myc-ALK2-R206H expressed alone or together with untagged ACVR2A or ACVR2B (one-way ANOVA and Bonferroni post hoc test). ( B ) Average R f values; ( C ) average D values. Bars depict the average values (mean ± SEM); the number of measurements is depicted within each bar. Asterisks indicate significant differences between the R f values of the pairs indicated by brackets (**, p < 8 × 10 −3 ; ****, p < 10 −4 ; one-way ANOVA and Bonferroni post hoc test. n.s. = not significant). Similar analysis of the D values showed no significant differences. The left panels in ( B , C ), designated “-Untag. Type II”, depict the R f and D values of myc-ALK2-R206H co-expressed with HA-ALK2-R206H without or with IgG αHA crosslinking; these values were taken from C,D and are shown to enable direct comparison with the effects of co-expressing untagged ACVR2A/B. Co-expression with untagged ACVR2A had no effect on ALK2-R206H homomeric interactions, which remained undetectable but were induced in the presence of ActA, conditions under which ACVR2A undergoes dimerization ( G). On the other hand, ACVR2B (which forms homodimers also without ActA; E) induced ALK2-R206H homomeric interactions already without ActA. The effects of kinase-dead untagged ACVR2B-KD were indistinguishable from those of ACVR2B.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Construct, Labeling, Expressing, Comparison

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ALK2-R206H-mediated pSMAD1/5/8 formation is induced by ACVR2B more efficiently than by ACVR2A. U2OS cells were transfected with vectors encoding HA-ALK2-R206H (or HA-ALK2-WT) alone or with myc-tagged ACVR2A, ACVR2B, or ACVR2B-KD. After 24 h, cells were starved (2 h, 1% serum) and stimulated (or not; control) with ActA (4 nM, 60 min, 37 °C). Cells were lysed, subjected to SDS--PAGE, and immunoblotted for pSMAD1/5/8, tSMAD1/5/8, and β-actin. As shown in , the cell-surface levels of the tagged receptors were similar and were not affected by the co-expressed receptors. ( A , B ) Representative blots of ActA signaling to pSMAD1/5/8. ( C , D ) Quantification of ActA-mediated pSMAD1/5/8 formation. The bands were visualized by ECL and quantified by densitometry. Data are mean ± SEM of the pSMAD1/5/8 over tSMAD1/5/8 ratio of 5 ( C ) or 4 ( D ) independent experiments. The value obtained for ActA-stimulated cells co-transfected with HA-ALK2-R206H and myc-ACVR2B was taken as 1. Asterisks show significant differences between the pairs indicated by brackets, using one-way ANOVA and Bonferroni post hoc test (*, p < 0.02; **, p < 4 × 10 −3 ; ***, p < 8 × 10 −4 ; n.s. = not significant).

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Transfection, SDS Page

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: ACVR2B is superior to ACVR2A in eliciting ALK2-R206H-mediated transcriptional activation of the SMAD1/5/8 pathway. U2OS cells were co-transfected with BRE-Luc and pRL-TK, together with HA-ALK2-R206H or HA-ALK2-WT (alone or together with myc-ACVR2A, myc-ACVR2B, or myc-ACVR2B-KD). These constructs were replaced by empty vector for control samples. After 17 h, cells were starved without serum (5 h) and stimulated (or not; control) with ActA (2 nM, 19 h). Relative Luminescence Units (RLU) are expressed as mean fold induction ± SEM (n = 4 independent experiments). The results were normalized for transfection efficiency using Renilla luminescence by the DLR luminescence assay. The value in untreated, unstimulated cells was taken as 1. The cell-surface levels of the tagged receptors were not altered by the co-expressed receptors . Asterisks show significant differences between the pairs indicated by the brackets, using one-way ANOVA and Bonferroni post hoc test (**, p < 1 × 10 −3 ; ***, p < 5 × 10 −4 ; ****, p < 10 −4 ; n.s. = not significant).

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Activation Assay, Transfection, Construct, Plasmid Preparation, Luminescence Assay

Journal: Cells

Article Title: The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A

doi: 10.3390/cells13030221

Figure Lengend Snippet: Model for the recruitment of ALK2-R206H into homomeric clusters by ACVR2A/B and its dependence on the dimeric nature of the type II receptors. ( A ) Homodimerization state of the singly-expressed receptors and the dependence on ActA. ACVR2B (red) forms stable homodimers already without ActA, which are enhanced by the ligand (thicker black arrow). ACVR2A (green) requires ActA (orange) to form homodimers. ALK2-WT (light blue) forms homodimers, while ALK2-R206H (dark blue) does not, and both are unaffected by ActA. ( B ) Effect of complex formation with ACVR2A or ACVR2B on the homomeric clustering of ALK2-R206H. The recruitment of the mainly monomeric ALK2-R206H into clusters by the type II receptor depends on the extent of homodimerization of the type II receptor. Thus, the largely dimeric ACVR2B can induce ALK2-R206H clustering already without ligand, while ActA enhances this effect due to increasing ACVR2B homomeric complex formation and its heteromeric interactions with ALK2-R206H. On the other hand, ACVR2A cannot induce clustering of ALK2-R206H, as both receptors are mainly monomeric in the absence of ligand. Upon binding of ActA, ACVR2A forms homodimers and can then induce clustering of ALK2-R206H. The homomeric clustering of ALK2-R206H leads to aberrant signaling to SMAD1/5/8 without a need for phosphorylation by the type II receptor.

Article Snippet: Human ALK2-WT with N-terminal HA tag in pCMV5 was described [ ], and N terminally myc-tagged human ALK2-WT in pCMV3 (cat. #HG14875-NM) was obtained from Sino Biological (Wayne, PA, USA).

Techniques: Binding Assay