rabbit anti-alk2 antibody  (Thermo Fisher)

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: Frontiers in Pharmacology

Article Title: BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling

doi: 10.3389/fphar.2024.1426121

Figure Lengend Snippet: Graphical representation of the experimental design. Experiment 1: Changes in pain sensitization and expressions of BMP10, ALK2, Smad1/5/8, phosphorylated Smad1/5/8, and GFAP after SNI in mice. Experiment 2: The effects of BMP10 siRNA on SNI-induced pain hypersensitivity and astrocytic activation. Experiment 3: The involvements of ALK2 in BMP10-induced pain hypersensitivity and astrocytic activation. Experiment 4: The effects of Smad1 siRNA on BMP10-induced pain hypersensitivity and astrocytic activation.

Article Snippet: The membranes were incubated with appropriate antibodies, including mouse anti-GFAP antibody (1:1,000, ab279289, Abcam), mouse anti-BMP10 antibody (1:1,000, 462732, Novus Biologicals, CO, United States), rabbit anti-ALK2 antibody (1:100, PA5-114818, Thermo Fisher Scientific, MA, United States), rabbit anti-phospho-Smad1/5/8 (1:1,000, AB3848-I, Sigma Aldrich, Germany), rabbit anti-Smad1/5/8 (1:1,000, NB100-56656, Novus Biologicals, CO, United States) and mouse anti-β-actin (1:1,000, AF2815, Beyotime Biotechnology , Shanghai, China) overnight at 4°C.

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling

doi: 10.3389/fphar.2024.1426121

Figure Lengend Snippet: Expression levels of ALK2, Samd1/5/8, and p-Smad1/5/8 in mouse ipsilateral spinal dorsal horn after SNI. (A–C) Western blot analysis showed the expression levels of ALK2, Samd1/5/8, and p-Smad1/5/8 in the ipsilateral (above) and contralateral (below) spinal dorsal horn of sham and SNI mice; (D) Double immunofluorescence staining showed the coexpression of ALK2 (red) with GFAP, Iba-1 and NeuN (green) in the ipsilateral spinal dorsal horn of SNI mice on postoperative Day 14 (scale bar = 50 μm/25 μm). Data are presented as mean and SEM; sham mice versus SNI mice on postoperative Days 7 and 14, *** p < 0.001 (n = 6).

Article Snippet: The membranes were incubated with appropriate antibodies, including mouse anti-GFAP antibody (1:1,000, ab279289, Abcam), mouse anti-BMP10 antibody (1:1,000, 462732, Novus Biologicals, CO, United States), rabbit anti-ALK2 antibody (1:100, PA5-114818, Thermo Fisher Scientific, MA, United States), rabbit anti-phospho-Smad1/5/8 (1:1,000, AB3848-I, Sigma Aldrich, Germany), rabbit anti-Smad1/5/8 (1:1,000, NB100-56656, Novus Biologicals, CO, United States) and mouse anti-β-actin (1:1,000, AF2815, Beyotime Biotechnology , Shanghai, China) overnight at 4°C.

Techniques: Expressing, Western Blot, Double Immunofluorescence Staining

Journal: Frontiers in Pharmacology

Article Title: BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling

doi: 10.3389/fphar.2024.1426121

Figure Lengend Snippet: ALK2 was involved in BMP10-induced pain hypersensitivity and astrocytic activation. (A, B) The time course of PWT and TWL in normal mice after intrathecal delivery of BMP10 peptide and reagents; Data are presented as mean and SEM, Vehicle group versus BMP10 group * p < 0.05, ** p < 0.01, *** p < 0.001; BMP10+ALK2-IN-2 group versus BMP group # p < 0.05, ## p < 0.01, ### p < 0.001 (n = 12); (C) Representative movement traces in OFT tests of mice after intrathecal delivery of BMP10 peptide and reagents; (D–F) Time in center zone, average speed and total distance of mice in open field after intrathecal delivery of BMP10 peptide and reagents; (G, H) immunofluorescence staining showed the expression of GFAP in the spinal dorsal horn after intrathecal delivery of BMP10 peptide and reagents (scale bar = 200/50 μm); (I) Western blot showed the expression of GFAP in the spinal cord after intrathecal delivery of BMP10 peptide and reagents; Data are presented as mean and SEM, Vehicle group versus BMP10 group * p < 0.05, ** p < 0.01, *** p < 0.001; BMP10+ ALK2-IN-2 group versus BMP group # p < 0.05, ## p < 0.01, ### p < 0.001 (n = 6).

Article Snippet: The membranes were incubated with appropriate antibodies, including mouse anti-GFAP antibody (1:1,000, ab279289, Abcam), mouse anti-BMP10 antibody (1:1,000, 462732, Novus Biologicals, CO, United States), rabbit anti-ALK2 antibody (1:100, PA5-114818, Thermo Fisher Scientific, MA, United States), rabbit anti-phospho-Smad1/5/8 (1:1,000, AB3848-I, Sigma Aldrich, Germany), rabbit anti-Smad1/5/8 (1:1,000, NB100-56656, Novus Biologicals, CO, United States) and mouse anti-β-actin (1:1,000, AF2815, Beyotime Biotechnology , Shanghai, China) overnight at 4°C.

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Western Blot

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 stabilizes ALK2 protein through direct binding. (a) After HEK293FT cells were co-transfected with the indicated plasmids, cells were immunoprecipitated (IP) with anti-HA antibody and subsequently immunoblotted (IB) with the anti-Flag or anti-HA antibody. (b) After HCT116 cells were treated with 100 ng/ml BMP6, endogenous PSMD14 was immunoprecipitated with anti-PSMD14 antibody and subsequently immunoblotted with the indicated antibodies against endogenous ALK2 and PSMD14 proteins. (c) Expression of endogenous ALK2 and ID3 proteins in PSMD14 -depleted or control (siCON) HCT116 cells were examined by immunoblot analysis (left). Expression of ALK2 mRNA in PSMD14 -depleted or control HCT116 cells was analyzed by qRT-PCR (right). (d) After a plasmid encoding HA-ALK2 was co-transfected into 293FT cells with dose-dependent expression of Flag-PSMD14, cells were immunoblotted with the indicated antibodies. (e) PSMD14-depleted or control (siCON) HCT116 cells were treated with 20 μM cycloheximide (CHX; protein synthesis inhibitor) for the indicated times. Cells were immunoblotted with anti-ALK2 and anti-PSMD14 antibodies. (f) After plasmids encoding wild-type (WT) Flag-PSMD14 and catalytic inactive DUB mutants of PSMD14 were respectively transfected into HCT116 cells, cells were treated with 20 μM CHX for the indicated times and immunoblotted. In (e) and (f) , ALK2 levels were quantified by using ImageJ software and normalized to β-actin expression. The data were statistically analyzed and the bars represent the mean ± s .d. from three independent experiments. * P <0.05, *** P < 0.001 (one-way ANOVA followed by Dunnett's test, n = 3, compared to the indicated controls). (g) PSMD14 -depleted and control (siCON) HCT116 cells were treated with 100 ng/ml BMP6 for 10 min and immunostained with the indicated antibodies to detect endogenous PSMD14 (green) and ALK2 (red). PKH26 molecule (yellow) and DAPI (blue) were used to stain the cell membrane and nuclei. Scale bar; 10 μm. (h) After HCT116 cells were treated with BMP6 for 10 min, cells were fractionated into membrane (Mem), cytoplasmic (Cyt) and nuclear (Nuc) extracts, which were subsequently immunoblotted with the indicated antibodies. Expressions of α-Tubulin, Lamin B1 and E-cadherin were used as markers and loading controls of cytosolic, nuclear and membrane fractions, respectively. Except for ( h ), expression of β-actin was used as a loading control of immunoblot analysis. The images in this figure are representative of three independent experiments.

Article Snippet: For immunohistochemistry, each TMA slide was stained with rabbit anti-PSMD14 antibody (HPA002114, 1:100; Sigma-Aldrich), rabbit anti-ALK2 antibody (PA5-13881, 1:100; Thermos Fisher Scientific).

Techniques: Binding Assay, Transfection, Immunoprecipitation, Expressing, Control, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Software, Staining, Membrane

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 stabilizes ALK2 protein through deubiquitinating Smurf1-mediated polyubiquitination of ALK2 (a, b) Plasmids encoding Flag-ALKs, Flag-PSMD14 or HA-Ubi were co-transfected into HEK293FT cells according to the indicated combinations. Ubiquitination of Flag-ALK2, Flag-ALK3, and Flag-ALK6 were examined by immunoprecipitation (IP) and immunoblots (IB) with the indicated antibodies. (c) Flag-ALK2 and Flag-PSMD14 were co-transfected into HEK293FT cells with a plasmid encoding wild-type or lysine mutant (K48 or K63) HA-Ubi in the indicated combinations. When the K48 mutant of His-Ubi was transfected, MG132 was treated to prevent ALK2 degradation. (d) The catalytically inactive DUB mutant of Flag-PSMD14 (H113Q, C120A or C120S) and wild-type Flag-PSMD14 were co-transfected into HEK293FT cells with Flag-ALK2 and HA-Ubi in the indicated combinations. (e) After PSMD14-depleted and control HCT116 cells were treated with 100 ng/ml BMP6 for the indicated times, ubiquitination of endogenous ALK2 protein was examined by IP and IB with the indicated antibodies. IgG was used as a negative control for IP. (f) After a plasmid encoding HA-ALK2 was co-transfected into HEK293FT cells with plasmids encoding Flag-Smurf1, Flag-Smurf2, Flag-CHIP, or Flag-TRIM33, respectively, co-immunoprecipitation assays were performed with the indicated antibodies. (g) Flag-Smurf1 or Flag-Smurf2 was co-transfected into HEK293FT cells with HA-Ubi and Flag-ALK2 in the indicated combinations. (h) Flag-ALK2 was co-transfected into HEK293FT cells with wild-type His-Ubi or a lysine mutant (K48 or K63) of HA-Ubi in the absence or presence of Flag-Smurf1. (i) Flag-Smurf1 and the K48 lysine mutant of HA-Ubi were co-transfected into HEK293FT cells with wild-type Flag-PSMD14 or the catalytic inactive DUB mutant of PSMD14 (H113Q). In (a) - (d) , ubiquitination of Flag-ALK proteins were examined by immunoprecipitation (IP) and immunoblots (IB) with the indicated antibodies. In (g) - (i) , ubiquitination of the ALK2 protein was analyzed by IP and IB with the indicated antibodies. In (h) and (i) , cells were pre-treated with MG132 to prevent protein degradation. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments.

Article Snippet: For immunohistochemistry, each TMA slide was stained with rabbit anti-PSMD14 antibody (HPA002114, 1:100; Sigma-Aldrich), rabbit anti-ALK2 antibody (PA5-13881, 1:100; Thermos Fisher Scientific).

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Plasmid Preparation, Mutagenesis, Control, Negative Control, Expressing

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 and ALK2 are required for BMP-mediated colon cancer tumorigenesis. (a) For the analysis of cell proliferation, 1 × 10 3 cells for each cell line were cultured in 12-well plates in the absence or presence of 100 ng/ ml BMP6 for the indicated times. Cell numbers were counted at the indicated time points. (b) BrdU incorporation assays were used to detect cell proliferation. (c) MTT assays were performed to analyze cell viability. (d) For colony forming assays, cells were seeded in 6-well plates with soft-agar media and incubated for 14 days. Colonies were counted and described as graphs demonstrating the percentage of colonies in the field. In (a) - (d) , the data were statistically analyzed and the error bras represent the mean ± s .d. * P <0.05, ** P <0.01, *** P < 0.001, ns; not significant (one-way ANOVA followed by Dunnett's test. n = 3 per group, compared to the indicated controls). (e) 1 × 10 6 PSMD14 -depleted, ALK2 -depleted or control HCT116 cells were injected in the NOD-SCID mouse group ( n = 5 per group). The tumorigenesis experiment was processed for 30 days. (f) Cell lysates isolated from each tumor xenograft were immunoblotted with the indicated antibodies. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments. (g) Tumor sizes ( n = 5 per group) were calculated every 5 days. (h) After isolating each tumor from mice ( n = 5 per group), tumor weights were measured and calculated as the average per group. In (g) and (h) , the data were statistically analyzed and the error bras represent the mean ± s .d. *** P < 0.001, ns; not significant (one-way ANOVA followed by Dunnett's test. n = 5 per group, compared to the indicated controls). In (a) - (h) , PSMD14 - and ALK2 -depleted HCT116 cells were generated by the infection of recombinant lentiviruses expressing shRNAs targeting ALK2 or PSMD14 .

Article Snippet: For immunohistochemistry, each TMA slide was stained with rabbit anti-PSMD14 antibody (HPA002114, 1:100; Sigma-Aldrich), rabbit anti-ALK2 antibody (PA5-13881, 1:100; Thermos Fisher Scientific).

Techniques: Cell Culture, BrdU Incorporation Assay, Incubation, Control, Injection, Isolation, Expressing, Western Blot, Generated, Infection, Recombinant

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: PSMD14 depletion reduces BMP6-mediated colorectal cancer stemness. (a, b) FACS analysis of CD133 + /CD44 + cells in PSMD14 - or ALK2 -depleted HCT116 cells, which were treated with 100 ng/ml BMP6 for 48 h. The proportion of the CD133 + /CD44 + fraction was described with the density plots (a) and a bar graph (b) . shGFP-expressing HCT116 cells were used as a control. (c) Sphere forming assay of PSMD14 - or ALK2 - depleted HCT116 cells. Spheres with a diameter above 50 μm were counted and described in a bar graph. Scale bars, 50 μm. (d) PSMD14 - or ALK2 -depleted HCT116 cells were treated with BMP6. Expression of pluripotent transcription factors were analyzed by immunoblotting with the indicated antibodies. siCON-expressing HCT116 cells were used as a control. (e, h) 2 × 10 4 cells of PSMD14 -, ALK2 -, ABCA7 - or ABCC4 -depleted HCT116 were respectively treated with 20 μM oxaliplatin and 30 μM DAPT and their viabilities were measured at 6 h. shGFP-expressing HCT116 cells were used as a control. (f, g) PSMD14 - or ALK2 -depleted HCT116 cells were treated with BMP6 for 6 h. Expressions of ABCA7 and ABCC4 were measured by quantitative RT-PCR. The data were statistically analyzed by two-way ANOVA followed by Bonferroni's multiple comparison test ( n = 3, *** P < 0.001 compared to the indicated controls. ns; not significant). The bars represent the mean ± s .d. The images in this figure are representative of three independent experiments. In (b), (c), (e) and (h) were statistically analyzed by one-way ANOVA followed by Dunnett's test ( n = 3, ** P <0.01, *** P < 0.001 compared to the indicated controls. ns; not significant).

Article Snippet: For immunohistochemistry, each TMA slide was stained with rabbit anti-PSMD14 antibody (HPA002114, 1:100; Sigma-Aldrich), rabbit anti-ALK2 antibody (PA5-13881, 1:100; Thermos Fisher Scientific).

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, Comparison

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: Higher expression of PSMD14 and ALK2 confer poor prognosis in human colorectal cancer. (a) Cell lysates isolated from the indicated colorectal cancer cell lines were immunoblotted with the ALK2 and PSMD14 antibodies. Expression of β-actin was used as a loading control. The images in immunoblot analyses are representative of three independent experiments. (b, c) Using the Kaplan-Meier (KM) plotter tool, expression of PSMD14 and ALK2 mRNA between normal and tumor tissues of colorectal cancer patients in a public GEO dataset (GSE21510; n = 148) were analyzed. (d) To verify the correlation of ALK2 mRNA expression in normal colon tissues and tumor tissues with the expression levels of PSMD14, a public GSE dataset (GSE21510) was analyzed by the KM plotter tool. In (b) - (d) , the boxes represent the interquartile range, centre is the median, and the minimum and maximum values are represented in the whiskers. *** P <0.001, ns; not significant (Student t -test, compared to normal tissues or PSMD14-low samples). (e) Correlation of ALK2 and PSMD14 mRNAs in normal colon tissues, polyps in colon, and colorectal tumor tissues of human colorectal cancer patient samples (GSE68468, n = 288). Samples were classified into three groups (double low expression of ALK2 and PSMD14 , single high expression of ALK2 or PSMD14 , and double high expression of ALK2 and PSMD14 ) in normal tissues, polyps and tumors. (f, g) Scatter dot plot analysis represents the scores of the expression of PSMD14 and ALK2 protein in the matched normal and tumor tissues of human colon cancer patients (normal tissue n = 70, cancer tissue n = 70) analyzed by immunohistochemistry. *** P <0.001 (Student t -test, compared to normal tissues). (h) Scatter dot plot shows Spearman correlations between ALK2 and PSMD14 protein expression according to scoring within all samples. *** P < 0.001 (Student t -test). The Spearman r indicates the Spearman correlation coefficients. (i, j) Correlation of PSMD14 or ALK2 mRNA expression with overall survival rates of human colorectal cancer patients were analyzed by a KM plot analysis in a public GSE dataset (GSE17538; n = 224 patients). P = 0.0372, P = 0.0021 (Log-rank test). HR = hazard ratio.

Article Snippet: For immunohistochemistry, each TMA slide was stained with rabbit anti-PSMD14 antibody (HPA002114, 1:100; Sigma-Aldrich), rabbit anti-ALK2 antibody (PA5-13881, 1:100; Thermos Fisher Scientific).

Techniques: Expressing, Isolation, Control, Western Blot, Immunohistochemistry

Journal: EBioMedicine

Article Title: The deubiquitinating enzyme PSMD14 facilitates tumor growth and chemoresistance through stabilizing the ALK2 receptor in the initiation of BMP6 signaling pathway

doi: 10.1016/j.ebiom.2019.10.039

Figure Lengend Snippet: Schematic representation of the proposed mechanism of BMP6 signaling pathway regulated by PSMD14-ALK2 axis in colorectal cancers. ALK2 type I receptor is polyubiquitinated by E3 ligase Smurf1 and subsequently degraded in the absence of BMP6. Upon treatment of BMP6, PSMD14 binds to ALK2 protein and deubiquitinates the K48-linked polyubiquitin chains of ALK2 protein, resulting in the increase of ALK2 stability and thus leads to the initiation of BMP6 signaling pathway. This initiation of BMP6 signaling pathway facilitates tumor growth, cancer stemness and chemoresistance in colorectal cancers.

Article Snippet: For immunohistochemistry, each TMA slide was stained with rabbit anti-PSMD14 antibody (HPA002114, 1:100; Sigma-Aldrich), rabbit anti-ALK2 antibody (PA5-13881, 1:100; Thermos Fisher Scientific).

Techniques:

Journal: Journal of Neuroscience

Article Title: Bone Morphogenetic Protein Signaling in the Developing Telencephalon Controls Formation of the Hippocampal Dentate Gyrus and Modifies Fear-Related Behavior

doi: 10.1523/jneurosci.0550-10.2010

Figure Lengend Snippet: Figure1. GeneratingdoublemutantmicedeficientinBmpr1aandBmpr1b.A–C,CoronalsectionsthroughthetelencephalonofcontrolmiceatE11.5,processedwithinsituhybridizationtoshow expression of Bmpr1a (A), Bmpr1b (B), and a third type I BMP receptor gene, Alk2 (C). Bmpr1a is expressed throughout the telencephalic neuroepithelium including the cortical hem; Bmpr1b is expressedmoreselectivelyinthedorsaltelencephalonwithahotspotofexpressionatthehem;Alk2isexpressedbroadlyinthedorsalandventraltelencephalon,butnotinthecorticalhem(C,C). D,BreedingstrategyusedtogeneratemicelackingBmpr1bconstitutivelyandBmpr1aconditionally,theBmpr1afx /;Bmpr1b /;Emx1 /IREScredoublemutantgenotype.E,PCRanalysisofDNA extracted from the tail and telencephalon of E12.5 Bmpr1afx /;Emx1 /IREScre and Bmpr1afx / mice. Primers fx1 and fx4 (Mishina et al., 2002) amplified a 180 bp fragment from Bmpr1afx /; Emx1 /IREScretelencephalon(lane4),indicativeofCre-mediatedrecombinationofBmpr1afx.The180bp“recombined”bandwasnotamplifiedfromtailtissue(lane2)orBmpr1afx /telencephalon (lane 6). Primers fx3 and fx5 amplified a 190 bp fragment from the Bmpr1a constitutive null allele in all three tissue samples (lanes 1, 3, 5). F, G, Coronal sections through the hem region at E12.5 inacontrolmouse(c)andadoublemutant(dm),immunostainedforpSmad1/5/8,transcriptionfactorsactivateddownstreamofBMPsignaling.TheredarrowsindicatepSmad1/5/8-IRcellsinthe controlhem(F)butvirtuallynopSmad1/5/8-IRcellsinthedoublemutanthem(G).Outsidethehem,inthehippocampalprimordium,pSmad1/5/8-IRcellsaredensealongtheventricularsurface (F,G).H,I,Ayoungadultcontrolmouse(H)andlittermatedoublemutant(I).Thedoublemutantisslightlysmallerthanthecontrol;theredarrowsindicatetruncateddigitsandpartiallossoffacial hair(seeResults).Abbreviations:hem,Corticalhem;hp,hippocampus;lge,lateralganglioniceminence;mge,medialganglioniceminence;ncx,neocortex;th,thalamus.Scalebar:(inA)A–C,200 m; C, 100 m; F, G, 50 m.

Article Snippet: For immunohistochemistry, primary antibodies were as follows: antiphospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/ 428) rabbit polyclonal antibody (1:50; Cell Signaling Technology), antiphosphohistone H3 (PH3) (Ser10) rabbit polyclonal antibody (1:200; Millipore), anti-calbindin D-28K rabbit polyclonal antibody (1:250; Millipore Bioscience Research Reagents), anti-neuronal nuclei (NeuN) mouse monoclonal antibody (1:10; Millipore), anti-5-bromo-2-deoxyuridine (BrdU) mouse monoclonal antibody (1:75; BD Biosciences) for IHC and anti-BrdU mouse monoclonal antibody (1:50; Serotec) for immunofluorescence, anti-tyrosine hydroxylase (TH) antibody (1:5000; BD Biosciences), anti- -tubulin (Tuj1) (1:500; Covance), anti-Axin2 rabbit polyclonal antibody (1:5000; Abcam), anti-Olig2 rabbit polyclonal antibody (1:1000; Abcam), anti-(cleaved) caspase 3 rabbit polyclonal antibody (1:200; Cell Signaling), anti-Dickkopf1 (DKK1) rabbit polyclonal antibody (1:2000; Santa Cruz Biotechnology), anti-calretinin rabbit polyclonal antibody (1:1000; Millipore), and anti-Acvr1 (ALK2) rabbit polyclonal antibody (1:2000; Novus Biologicals).

Techniques: Expressing, Amplification