human alk2 proteins  (Sino Biological)

Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: A Flow cytometry analysis in ATDC5 cells overexpressing ALK2 WT or mutant ALK2 R206H receptors in regular medium (5% FCS) and serum starvation (starvation) conditions. GFP-positive and RFP-positive cells were detected by fluorescence emitted under 488 nm and 561 nm excitation lasers, respectively. Representative histograms of a single experiment with median fluorescence intensity of GFP and RFP are shown. Cumulative plots of six independent experiments show mean ± SD of GFP/RFP fluorescence ratio in serum starvation conditions versus regular medium (DMEM F12 5% FBS). Statistical analysis: One-way ANOVA (Tukey’s multiple comparison) test was performed (ns = not significant; ** P ≤ 0.01; **** P ≤ 0.0001) ( B ) Representative immunoblotting and relative densitometric analysis ( n = 3) on total protein extracts derived from ATDC5 cells grown in regular medium (5% FBS) or in serum starvation condition (starvation). Tubulin and HSP90 were used as loading control. Statistical analysis: unpaired t -test (ns = not significant; ** P ≤ 0.01; **** P ≤ 0.0001) ( C ) Confocal quantification of the number of autophagic vesicles in ATDC5 cells in starvation condition after loading with 50 µM MDC. Data analysis of MDC spot number was performed using the Harmony software (ver 4.5) of the Opera Phenix high-content system. Scale bar, 10 μm. Statistical analysis: unpaired t- test (**** P ≤ 0.0001).

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Flow Cytometry, Mutagenesis, Fluorescence, Comparison, Western Blot, Derivative Assay, Control, Software

Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: A Histograms show the mRNA expression for the indicated genes expressed as fold increase (mean ± SD) from three independent experiments in ATDC5 cells overexpressing ALK2 WT or mutant ALK2 R206H in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h). GAPDH mRNA was used to normalize data. B Immunoblotting and corresponding densitometric analysis with the indicated antibodies of ATDC5 cells overexpressing ALK2 WT or mutant ALK2 R206H in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h) ( C ) Histograms show the mRNA expression for the indicated gene expressed as fold increase (mean ± SD) from three independent experiments in ATDC5 cells overexpressing ALK2 WT or mutant ALK2 R206H in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h). GAPDH mRNA was used to normalize data. D , E Immunoblotting and relative densitometric analysis with the indicated antibodies of U2OS cells overexpressing ALK2 WT or mutant ALK2 R206H DDK tagged, in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h) and treated, as indicated, with 20 μM CQ (2 h). F Confocal microscopy analysis using anti-DDK (red) in U2OS cells expressing ALK2 WT or mutant ALK2 R206H DDK tagged receptors treated or not, as indicated, with chloroquine (CQ, 20 μM, 2 h) in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h). DNA was counterstained with DAPI (blue). Scale bar 10 µm. DDK fluorescence intensity was measured by using Fiji software (ImageJ). At least 50 cells from 3 independent experiments were analyzed unpaired t -test (Welch’s correction) was performed. Data are presented as mean ± SD of at least three independent experiments. Symbols (dots) represent individual cells. Statistical analysis: unpaired (ns = not significant; * P ≤ 0.05; **P ≤ 0.01; *** P < 0.001 ****P ≤ 0.0001).

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Expressing, Mutagenesis, Western Blot, Confocal Microscopy, Fluorescence, Software

Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: A Representative images and relative quantification of EGFP-LC3-labeled autophagosomes colocalization with ALK2-DDK receptor. U2OS EGFP-LC3 cells expressing ALK2 WT or mutant ALK2 R206H DDK tagged receptors were treated with chloroquine (CQ, 20 μM, 2 h) in hypoxic conditions (1% O 2 ,16 h). Images were acquired by spinning disk confocal microscopy and analyzed for colocalization of EGFP-LC3 (green) and DDK (red) dots using the ComeDet plugin of the Fiji ImageJ software. Data are presented as mean ± SD and normalized on total EGFP-LC3 dots. DNA was counterstained with DAPI (blue). Insets show a 3-fold enlargement of the boxed areas. Scale bar, 10 μm is shown. At least 50 cells from 3 independent experiments were analyzed unpaired t -test (Welch’s correction) was performed. (ns = not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P < 0.001 **** P ≤ 0.0001). B Immunoblotting and relative densitometric analysis, numbers report the densitometric values of band intensity, with the indicated antibodies of U2OS cells overexpressing ALK2 WT or mutant ALK2 R206H in normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ,16 h) and stably infected with lentiviral construct expressing shATG5 RNA interference or expressing an empty vector as control (shCTRL).

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Labeling, Expressing, Mutagenesis, Confocal Microscopy, Software, Western Blot, Stable Transfection, Infection, Construct, Plasmid Preparation, Control

Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: A Immunoblotting and relative densitometric analysis with the indicated antibodies of ALK2 R206H -DDK U2OS cells treated or not, at different time points, with activin A (100 ng/ml) and Rapamycin (Rapa, 100 ng/ml) in serum starvation conditions. Histograms show the mean ± SD of at least three independent experiments. Statistical analysis: unpaired t -test (ns = not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 **** P ≤ 0.0001). B Representative fluorescence images of U2OS cells expressing mutant ALK2 R206H DDK tagged receptors treated or not, as indicated, with Rapamycin (Rapa, 100 ng/ml, 5 h) and/or chloroquine (CQ, 20 μM, 2 h), immunostained with anti-LAMP2 (red) and anti-DDK (green) antibodies and counterstained with DNA (DAPI, blue). Scale bar 10 µm. Images were acquired by spinning disk confocal microscopy and analyzed for DDK fluorescence intensity using the Fiji ImageJ software. Data are presented as median with interquartile range. N ≥ 20 cells per sample from two independent experiments. Symbols represent individual cells. Insets show a 3-fold enlargement of the boxed areas. Statistical analysis: one-way ANOVA (Tukey’s multiple comparison) test was performed (** P ≤ 0.01; **** P ≤ 0.0001). C Flow cytometry analysis of ALK2 R206H ATDC5 cells cultured in serum starvation conditions, left untreated (NT) or treated with activin A (100 ng /ml) alone (DMSO), or in combination with Chloroquine (CQ, 20 µM, 2 h), Rapamycin (Rapa, 100 ng/ml, 24 h) for 24 h. Representative histograms show the median fluorescence intensity (MFI) of fluorescence GFP and RFP emitted as a result of excitation with blue (488 nm) and yellow (561 nm) lasers, respectively. Cumulative plots report the mean ± SD of GFP/RFP fluorescence ratio versus non-treated (NT) cells ( n = 3). Statistical analysis: one-way ANOVA (Tukey’s multiple comparison) test was performed (ns = not significant; ** P ≤ 0.01).

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Western Blot, Fluorescence, Expressing, Mutagenesis, Confocal Microscopy, Software, Comparison, Flow Cytometry, Cell Culture

Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: A Chondrogenic differentiation micromass assay in ATDC5 ALK2 R206H cells and in ATDC5 ALK2 WT cells. Cells were incubated for 21 days in differentiation medium containing or not (NT), activin A (100 ng/ml) and treated, as indicated, with autophagy inhibitors Chloroquine (CQ, 20 μM), 3-Methyladenine (3-MA, 20 μM) or autophagy inducers Rapamycin (Rapa, 100 ng/ml), Spermidine (SPD, 20 μM) or DMSO. Representative images of Alcian blue staining. Histograms representing Alcian blue quantification measured by absorbance at 595 nm after solubilization with guanidine hydrochloride. All results represented the mean ± SD of at least three independent experiments. Statistical analysis: One-way ANOVA (Tukey’s multiple comparison) test was performed (** P ≤ 0.01; *** P < 0.001 **** P ≤ 0.0001). B Real-time PCR of Col10a1 mRNA in ATDC5 cells treated or not with activin A for 21 days in micromass cultures. mRplp0 was used to normalize data. All results represented the mean ± SD of at least three independent experiments. Statistical analysis: unpaired t- test (** P ≤ 0.01; *** P < 0.001 **** P ≤ 0.0001). C Histogram showing measurement of ALP activity in ATDC5 cells treated or not as indicated for 5 days in micromass culture. 5 × 10 4 cells were homogenized in 1 ml of assay Buffer, diluted 1:10 in assay Buffer, and 80 μl was used to measure ALP activity. Assays were performed following the fluorimetric kit protocol (MAK411, Sigma–Aldrich) and measured at O.D. 405 nm reading with Varioskan LUX Plate Reader. All results represented the mean ± SD of at least three independent experiments. Statistical analysis: A one-way ANOVA (Tukey’s multiple comparison) test was performed (** P ≤ 0.01; *** P < 0.001). Chondrogenic differentiation micromass assay in ATDC5 ALK2 R206H cells stable expressing shRNA for ATG4 (shATG4) ( D ) or ATDC5 ALK2 WT and ALK2 R206H cells stable expressing shRNA for Rubicon (shRubicon) ( E ), upon lentiviral infection. A lentiviral empty vector was used as control (shCTRL). Cells were incubated for 21 days in differentiation medium containing or not (NT), activin A (100 ng/ml) and treated as indicated with Rapamycin (Rapa 100 ng/ml) or DMSO. Representative images of Alcian blue staining and relative histograms showing Alcian blue quantification measured by absorbance at 595 nm after solubilization with guanidine hydrochloride. All results represented the mean ± SD of at least three independent experiments for ( D ) and from six independent experiments for ( E ). Statistical analysis: One-way ANOVA (Tukey’s multiple comparison) test was performed (* P ≤ 0.05; ** P ≤ 0.01, **** P ≤ 0.0001).

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Incubation, Staining, Comparison, Real-time Polymerase Chain Reaction, Activity Assay, Expressing, shRNA, Infection, Plasmid Preparation, Control

Journal: Cell Death Discovery

Article Title: Interplay between ALK2 R206H mutant receptor and autophagy signaling regulates receptor stability and its chondrogenic functions

doi: 10.1038/s41420-025-02393-0

Figure Lengend Snippet: Schematic illustration showing a simplified model of the interplay between the mutant ALK2 R206H receptor (without showing the type I/type II receptors tetramer) and the mTOR/autophagy signaling in the regulation of the balance between receptor degradation and chondrogenesis upon hypoxic conditions or activin A stimulation.

Article Snippet: For immunoblotting, 20–30 μg of protein extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted on nitrocellulose membrane, and stained with the specific primary antibodies: anti-Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E,Cell Signaling Technology), anti-p62 (SQSTM1) (PM045; MBL International), anti-GAPDH (D16H11) (Cell Signaling Technology), anti-LC3B (Sigma–Aldrich, L7543), anti-NanoLuc (Promega Corporation, N700A), anti-DDK (FLAG) (Origene, TA50011-100), anti ALK2/ACVR1 (SinoBiological.

Techniques: Mutagenesis

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