Journal: The Journal of endocrinology

Article Title: Activin A and activin receptors in gestational tissue from preeclamptic pregnancies.

doi: 10.1677/joe.0.1710057

Figure Lengend Snippet: Figure 3 Localisation of activin A subunit and receptor Alk2. Panels A and B show activin A staining in healthy term and preeclamptic term placentae respectively. Panel B shows intense staining in syncytiotrophoblast (st) of preeclamptic placenta. Less intense staining was seen in cytotrophoblast (ct), and stroma (s) of healthy and preeclamptic placentae. Fetal membranes from healthy and preeclamptic pregnancies are shown in panels C and D respectively. A staining is present in amniotic epithelium (ep), fibroblast layer (fib), reticular layer (r), chorionic trophoblast (t) and adherent decidua (dec). Receptor Alk2 staining in healthy and preeclamptic placentae is seen mainly in the endothelium (en) of the placental vasculature (panels G and H respectively). The insert in panel G is a serial section with the vasculature stained with the endothelial marker CD34. Alk2 staining in healthy and preeclamptic membranes (panels I and J respectively) is similar to A except that the amniotic epithelium and chorionic trophoblast were mostly negative. Positive and negative control sections (endometrium) for A are shown in panels E and F and panels K and L for Alk2. Scale bars=50 m except panels E, F, K and L where scale bars=25 m.

Article Snippet: Non-specific binding was blocked with 1% gelatine and 0·02% Tween 20 in Tris-buffered saline and blots were incubated with polyclonal goat anti-human activin receptor Alk2, ActRII and ActRIIB antibodies (0·3 μg/ml; R & D Systems, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Staining, Marker, Negative Control

Journal: The Journal of endocrinology

Article Title: Activin A and activin receptors in gestational tissue from preeclamptic pregnancies.

doi: 10.1677/joe.0.1710057

Figure Lengend Snippet: Figure 4 Western hybridisation; representative blots with placental, choriodecidual and amnion tissue protein (upper, mid and lower panels respectively) hybridised with activin receptor Alk2, ActRII and ActRIIB antibodies. Each blot contains proteins from T-C, PT-PE, T-PE and endometrium (E, positive control).

Article Snippet: Non-specific binding was blocked with 1% gelatine and 0·02% Tween 20 in Tris-buffered saline and blots were incubated with polyclonal goat anti-human activin receptor Alk2, ActRII and ActRIIB antibodies (0·3 μg/ml; R & D Systems, Minneapolis, MN, USA) for 1 h at room temperature.

Techniques: Western Blot, Hybridization, Positive Control

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

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 immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

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 immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

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 immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

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 immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

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 immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

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 immunofluorescence assays, PKH26 (PKH26GL, 1:1000; Sigma-Aldrich) was administered to cells for 10 min and cold methanol at −20 °C was used to fix cells for 10 min, and subsequently, 5% BSA in PBS solution was used for blocking at room temperature for 30 min. Next, samples were incubated with primary antibodies at 4 °C for 15 h. Mouse polyclonal anti-PSMD14 antibody (SAB1408741, 1:100; Sigma-Aldrich), rabbit polyclonal ALK2 antibody (orb314588, 1:100; Biorbyt, San Francisco, USA) were used to detect endogenous PSMD14 and ALK2, respectively.

Techniques:

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