Journal: Journal of Zhejiang University. Science. B

Article Title: Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats

doi: 10.1631/jzus.B2100366

Figure Lengend Snippet: Real-time PCR primer details

Article Snippet: The membranes were incubated with primary antibodies, including anti-CTSD (1:2000; Bioss, bs-1615R, Beijing, China), anti-BMPR-IB (1:1500; Bioss, bs-6639R), anti-FSHR (1:1500; Bioss, bs-0895R), anti-INHA (1:1000, Bioss, bs-1032R), and anti-‍β‍-‍actin (1:3000; Affinity Biosciences, AF7018, USA) ( Tang et al., 2019 ).

Techniques: Real-time Polymerase Chain Reaction, Sequencing, Amplification

Journal: Journal of Cell Science

Article Title: Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

doi: 10.1242/jcs.197285

Figure Lengend Snippet: Overexpression of the Cx43 CT domain competes for binding of signaling proteins with the intact Cx43 protein. Co-immunoprecipitations (co-IPs) were performed to assess protein–protein interactions between the indicated signaling proteins and the Cx43 C-terminus. UMR106 cells were transfected with a construct encoding the CT domain of Cx43 (FLAG-tagged) or with an empty vector control. (A) Input and bead fractions are shown for co-IPs performed with anti-ERK1/2, anti-PKCδ and anti β-catenin antibodies and blotted with anti-Cx43 antibodies. A negative control co-IP with an antibody to a protein unlikely to be found at the plasma membrane (anti-Sp1) was performed and blotted with anti-Cx43 antibodies and is shown below. (B) IPs were performed for the Cx43 C-terminus (with anti-FLAG antibody), and then blotted for the indicated signaling proteins.

Article Snippet: The FLAG-tagged Cx43 CT construct, pCMV3XFLAGm43CT (amino acids 236–382), was a gift from Cecilia Lo (Addgene plasmid # 17664).

Techniques: Over Expression, Binding Assay, Transfection, Construct, Plasmid Preparation, Negative Control, Co-Immunoprecipitation Assay

Journal: Journal of Cell Science

Article Title: Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

doi: 10.1242/jcs.197285

Figure Lengend Snippet: No apparent morphological defects are observed in mice with truncation of the Cx43 CT. (A) Digital X-rays, and (B) body length and weight measurements in 6-week-old male Gja1+/− (n=11) and Gja1−/K258Stop (n=8) mice. (B) Schematic of full-length (WT Cx43) and truncated Cx43 (Cx43 K258Stop) proteins, showing deletion of most of the C-terminus. (C) Western blot performed on tibial extracts (marrow flushed) for Cx43 (C-terminal epitope). qRT-PCR from RNA isolated from tibia (marrow flushed) of the indicated genotype was performed for Gja1 (Cx43; 3′ primers to the C-terminal encoding region) and Gjc1 (Cx45) mRNA expression. (n=7 for Gja1+/− and n=5 for Gja1−/K258Stop). Graphs depict mean±s.d. *P<0.05; n.s., not significant (two-tailed t-test).

Article Snippet: The FLAG-tagged Cx43 CT construct, pCMV3XFLAGm43CT (amino acids 236–382), was a gift from Cecilia Lo (Addgene plasmid # 17664).

Techniques: Western Blot, Quantitative RT-PCR, Isolation, Expressing, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

doi: 10.1242/jcs.197285

Figure Lengend Snippet: Truncation of Cx43 results in a cortical phenotype with increased cross-sectional area, increased cortical porosity, cortical thinning and marrow cavity expansion in 6-week-old male Gja1−/K258Stop mice. (A) Top, cross section through the femoral mid-diaphysis from a representative microCT slice from Gja1+/− and Gja1−/K258Stop mice. Bottom, 3D reconstruction of intact femurs. (B) Quantification of the cortical phenotype at the femoral mid-diaphysis by microCT (n=11 for Gja1+/− and n=8 for Gja1−/K258Stop); see Materials and Methods for the definitions of the parameters. Graphs depict mean±s.d. *P<0.05 (two-tailed t-test).

Article Snippet: The FLAG-tagged Cx43 CT construct, pCMV3XFLAGm43CT (amino acids 236–382), was a gift from Cecilia Lo (Addgene plasmid # 17664).

Techniques: Two Tailed Test

Journal: Journal of Cell Science

Article Title: Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

doi: 10.1242/jcs.197285

Figure Lengend Snippet: Cx43 truncation affects regulators of osteoclastogenesis, leading to increased osteoclast number and increased bone resorption. (A) qRT-PCR from RNA isolated from tibia (marrow flushed) of the indicated genotype was performed for RANKL and OPG mRNA expression (n=7 for Gja1+/− and n=5 for Gja1−/K258Stop). (B) Static histomorphometry of osteoclast number normalized to the bone surface area (OC.N/BS; n=5 per genotype). (C) Quantification of serum levels of CTX (n=3 for Gja1+/− and n=4 for Gja1−/K258Stop). Graphs depict mean±s.d. *P<0.05; n.s., not significant (two-tailed t-test).

Article Snippet: The FLAG-tagged Cx43 CT construct, pCMV3XFLAGm43CT (amino acids 236–382), was a gift from Cecilia Lo (Addgene plasmid # 17664).

Techniques: Quantitative RT-PCR, Isolation, Expressing, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

doi: 10.1242/jcs.197285

Figure Lengend Snippet: Cx43 truncation impairs signal cascade activation and osteoblast differentiation. (A) Western blots were probed for indicated signaling proteins in tibial (marrow flushed) extracts of the indicated genotypes. The GAPDH blot shown in Fig. 6A (right panel) is the same as in Fig. 8B, as the same membrane was re-probed for the indicated factors. qRT-PCR from RNA isolated from tibia (marrow flushed) of the indicated genotype was performed for (B) Wnt/β-catenin-related genes, (C) transcriptional regulators of osteoblastogenesis, and (D) osteoblast differentiation markers (n=7 for Gja1+/− and n=5 for Gja1−/K258Stop). (C) Western blots on extracts from BMSCs cultured for 7 days in mineralization medium were probed for Runx2, Osterix and GAPDH (loading control). Graphs depict mean±s.d. *P<0.05; n.s., not significant (two-tailed t-test).

Article Snippet: The FLAG-tagged Cx43 CT construct, pCMV3XFLAGm43CT (amino acids 236–382), was a gift from Cecilia Lo (Addgene plasmid # 17664).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Isolation, Cell Culture, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

doi: 10.1242/jcs.197285

Figure Lengend Snippet: Cx43 truncation reduces mRNA expression of collagen processing factors and results in a disorganized and less mature collagen network in cortical bone. (A) qRT-PCR from RNA isolated from tibia (marrow flushed) of the indicated genotype was performed for expression of collagen-processing factors. Graphs depict mean±s.d. (n=7 for Gja1+/− and n=5 for Gja1−/K258Stop). *P<0.05 (two-tailed t-test). Western blot on extracts from BMSCs cultured for 7 days in mineralization medium and immunoblotted for Lox, Hsp47 and GAPDH (load control) are also shown. Bands for the preform (pro) and mature isoform of Lox are detected. (B) Picrosirius Red staining was performed and quantified at the mid-diaphyseal cortical bone, using a hue-range distribution method. A representative image of mid-diaphyseal cortical bone is shown. Quantification of fiber orientation and length is shown as a percentage of composition of the birefringent hue (red, orange, yellow and green).

Article Snippet: The FLAG-tagged Cx43 CT construct, pCMV3XFLAGm43CT (amino acids 236–382), was a gift from Cecilia Lo (Addgene plasmid # 17664).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Two Tailed Test, Western Blot, Cell Culture, Staining

Journal: Journal of Cell Science

Article Title: Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

doi: 10.1242/jcs.197285

Figure Lengend Snippet: Truncation of Cx43 leads to cell autonomous defects in osteoblast proliferation, signaling and mineralization. (A) Cell proliferation on BMSCs after 48 h in culture (n=6 wells per genotype). (B) Western blot on extracts from BMSCs cultured for 7 days in mineralization medium and immunoblotted for indicated signaling proteins. The blot for GAPDH shown in Fig. 8B is the same as in Fig. 6A (right panel), as the same membrane was re-probed for the indicated factors. (C) Alizarin Red S staining in replicate wells (n>4 wells per genotype) in BMSCs cultured for 14 days in mineralization medium. Graphs depict mean±s.d. *P<0.05 (two-tailed t-test).

Article Snippet: The FLAG-tagged Cx43 CT construct, pCMV3XFLAGm43CT (amino acids 236–382), was a gift from Cecilia Lo (Addgene plasmid # 17664).

Techniques: Western Blot, Cell Culture, Staining, Two Tailed Test

Journal: Journal of Translational Medicine

Article Title: Organizing pneumonia in mice and men

doi: 10.1186/s12967-016-0933-6

Figure Lengend Snippet: Description of inflammation- and fibrosis-associated genes investigated in human (OP and control) lungs and in transgenic and nontransgenic mice

Article Snippet: BMPR1B , Abnova , 1:500.

Techniques: Transgenic Assay, Ligand Binding Assay, Activity Assay, Activation Assay, Produced, Migration, Expressing, Binding Assay, Cell Differentiation

Journal: Journal of Translational Medicine

Article Title: Organizing pneumonia in mice and men

doi: 10.1186/s12967-016-0933-6

Figure Lengend Snippet: Antibodies, source and dilutions used for immunohistochemistry

Article Snippet: BMPR1B , Abnova , 1:500.

Techniques:

Journal: Journal of Translational Medicine

Article Title: Organizing pneumonia in mice and men

doi: 10.1186/s12967-016-0933-6

Figure Lengend Snippet: Gene expression levels of the Tgfb1/TGFB1 cascade in murine OP lesions, compared to controls. TGFB1/Tgfb1, CXCL12/Cxcl12, TIMP1/Timp1, TIMP2/Timp2, COL3A1/Col3a1 and MMP2/Mmp2 show an OP lesion-specific overexpression in both human and murine lungs. In contrast, BMP4 and BMPR1B were upregulated in human OP lesions, compared to controls, while in murine lungs both genes were downregulated. There was no significant difference in the expression level of IL6/Il6. Ctrl.—control(s)

Article Snippet: BMPR1B , Abnova , 1:500.

Techniques: Expressing, Over Expression

Journal: Journal of Translational Medicine

Article Title: Organizing pneumonia in mice and men

doi: 10.1186/s12967-016-0933-6

Figure Lengend Snippet: Gene expression levels of the Tgfb1/TGFB1 cascade in human OP lesions, compared to controls. TGFB1/Tgfb1, CXCL12/Cxcl12, TIMP1/Timp1, TIMP2/Timp2, COL3A1/Col3a1 and MMP2/Mmp2 show an OP lesion-specific overexpression in both human and murine lungs. In contrast, BMP4 and BMPR1B were upregulated in human OP lesions, compared to controls, while in murine lungs both genes were downregulated. There was no significant difference in the expression level of IL6/Il6. Ctrl.—control(s)

Article Snippet: BMPR1B , Abnova , 1:500.

Techniques: Expressing, Over Expression

Journal: Developmental neuroscience

Article Title: Targeted Knockdown of Bone Morphogenetic Protein Signaling within Neural Progenitors Protects the Brain and Improves Motor Function following Postnatal Hypoxia-Ischemia

doi: 10.1159/000485379

Figure Lengend Snippet: Bone morphogenic protein receptor (BMPR) levels remain relatively stable after hypoxia-ischemia (HI) injury. Bar graphs representing quantification of BMPR1a (a), BMPR1b (b), and BMPR2 transcripts (c) following sham or HI surgery as determined by quantitative real-time polymerase chain reaction. y-axis represents change in threshold cycle (ΔC T), x-axis indicates time after surgery. *p = 0.02. d–f Bar graphs represent relative levels of BMPR1a (d), BMPR1b (e), and BMPR2 (f) protein in total cerebral lysates from sham or HI surgery brains. Quantification is band intensity relative to sham at 24 h. Representative blots are shown below each graph. n = 3 for each time point and treatment group. Error bars represent SEM.

Article Snippet: The following antibodies were used: CNPase 1: 1,000 (Sternberger SMI91), GAPDH 1: 1,000 (Millipore MAB374), MBP 1: 1,000 (Sternberger SMI99), phosphorylated SMAD1/5/8 (pSMAD1/5/8) 1: 1,000 (Cell Signaling #9511), BMPR1a 1: 100 (Abgent AP2004a), BMPR1b 1: 1,000 (Abgent AP2005c), and BMPR2 1: 40 (Abgent AP2006a).

Techniques: Real-time Polymerase Chain Reaction

Journal: Developmental neuroscience

Article Title: Targeted Knockdown of Bone Morphogenetic Protein Signaling within Neural Progenitors Protects the Brain and Improves Motor Function following Postnatal Hypoxia-Ischemia

doi: 10.1159/000485379

Figure Lengend Snippet: Bone morphogenic protein receptor 2 (BMPR2) knockdown in NG2 cells following injury decreases ventriculomegaly. Magnetic resonance images 42 dpl showing ipsilateral lateral ventricle (red), contralateral lateral ventricle (yellow), and third ventricle (blue). a, a ′, a ″ Three different brains from lesioned, vehicle-treated mice (LesVeh). b, b ′, b ″, b ‴ Four different brains from lesioned, tamoxifen-treated mice (LesTam). c Quantification of lateral ventricle volumes. Error bars represent SEM. contra, contralateral lateral ventricle; ispi, ipsilateral lateral ventricle. Four LesTam-ipsilateral, 4 LesTam-contralateral, 3 LesVeh-ipsilateral, and 3 LesVeh-contralateral ventricles. *p = 0.01, **p = 0.01, ***p = 0.01. d Quantification of 3rd ventricle volumes. Three LesVeh and 4 LesTam. Error bars represent SEM.

Article Snippet: The following antibodies were used: CNPase 1: 1,000 (Sternberger SMI91), GAPDH 1: 1,000 (Millipore MAB374), MBP 1: 1,000 (Sternberger SMI99), phosphorylated SMAD1/5/8 (pSMAD1/5/8) 1: 1,000 (Cell Signaling #9511), BMPR1a 1: 100 (Abgent AP2004a), BMPR1b 1: 1,000 (Abgent AP2005c), and BMPR2 1: 40 (Abgent AP2006a).

Techniques:

Journal: Developmental neuroscience

Article Title: Targeted Knockdown of Bone Morphogenetic Protein Signaling within Neural Progenitors Protects the Brain and Improves Motor Function following Postnatal Hypoxia-Ischemia

doi: 10.1159/000485379

Figure Lengend Snippet: Bone morphogenic protein receptor 2 (BMPR2) knockdown in NG2 cells following injury improves motor function. Representative left forelimb gait tracings for a nonlesioned, vehicle-treated mouse (ShamVeh) (a), a lesioned, vehicle-treated mouse (LesVeh) (b), and a lesioned, tamoxifen-treated mouse (LesTam) (c) at 14 dpl are shown. The height of each peak is the area of the footprint at peak stance. The number of peaks is the number of steps the animal takes within the time shown. d Quantification of mean paw area at peak stance. Error bars represent SEM. L, left; R, right. *p = 0.04, **p = 0.02, ***p = 0.02. e Quantification of paw area variability at peak stance for the left forelimb. *p = 0.03. f Quantification of maximum change in paw area per change in time (Max. dA/dT) for the left forelimb. *p = 0.03. g Ataxia coefficient for the left forelimb. Sample sizes were as follows: 3 sham, 8 LesVeh, and 6 LesTam. Error bars represent SEM. *p = 0.02, **p = 0.01. h RotaRod testing of 2-month-old uninjured vehicle-treated mice (n = 7) compared to tamoxifen-treated mice (n = 5). T1–T3, training days 1–3; A1/A2, acceleration days 1/2.

Article Snippet: The following antibodies were used: CNPase 1: 1,000 (Sternberger SMI91), GAPDH 1: 1,000 (Millipore MAB374), MBP 1: 1,000 (Sternberger SMI99), phosphorylated SMAD1/5/8 (pSMAD1/5/8) 1: 1,000 (Cell Signaling #9511), BMPR1a 1: 100 (Abgent AP2004a), BMPR1b 1: 1,000 (Abgent AP2005c), and BMPR2 1: 40 (Abgent AP2006a).

Techniques:

Journal: Developmental neuroscience

Article Title: Targeted Knockdown of Bone Morphogenetic Protein Signaling within Neural Progenitors Protects the Brain and Improves Motor Function following Postnatal Hypoxia-Ischemia

doi: 10.1159/000485379

Figure Lengend Snippet: Bone morphogenic protein receptor 2 (BMPR2) knockdown in NG2 cells following injury partially protects against myelin loss. Veh, vehicle; Tam, tamoxifen. n = 4, respectively. a Quantification of CNPase expression by mean relative immunofluorescence. Error bars represent SEM. b Quantification of MBP expression by mean relative immunofluorescence. *p = 0.02. c, d Representative micrographs of MBP expression within the corpus callosum 14 dpl. ×40. Scale bar, 25 μm. e, f Representative micrographs of Luxol fast blue (LFB) staining within the corpus callosum 14 dpl. Pink is cresyl violet. ×20. Scale bar, 50 μm. g, h Quantification of CNPase (g) and MBP expression (h) by Western blot of the ipsilateral internal capsule and representative blots. Four nonlesioned, vehicle-treated mice (LesVeh) and 5 lesioned, tamoxifen-treated mice (LesTam). Error bars represent SEM. a.u., arbitrary units.

Article Snippet: The following antibodies were used: CNPase 1: 1,000 (Sternberger SMI91), GAPDH 1: 1,000 (Millipore MAB374), MBP 1: 1,000 (Sternberger SMI99), phosphorylated SMAD1/5/8 (pSMAD1/5/8) 1: 1,000 (Cell Signaling #9511), BMPR1a 1: 100 (Abgent AP2004a), BMPR1b 1: 1,000 (Abgent AP2005c), and BMPR2 1: 40 (Abgent AP2006a).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot

Journal: Developmental neuroscience

Article Title: Targeted Knockdown of Bone Morphogenetic Protein Signaling within Neural Progenitors Protects the Brain and Improves Motor Function following Postnatal Hypoxia-Ischemia

doi: 10.1159/000485379

Figure Lengend Snippet: Bone morphogenic protein receptor 2 (BMPR2) knockdown in NG2 cells following injury does not affect oligodendroglial lineage. Representative confocal micrographs of cells within the corpus callosum 7 dpl. Markers used are indicated on the left of each set of panels. ShamVeh, sham surgery, vehicle-treated; LesVeh, hypoxia-ischemia (HI) surgery, vehicle treated; LesTam, HI surgery, tamoxifen treated. Antibodies tested were Olig1 (a, a′, a ″), Olig2 (c, c ′, c″), PDGFRα (e, e ′, e″), CC1 (h, h′, h″), GFAP (j, j′, j″), and Iba1 (l, l ′, l″). b, d, f, g, i, k, m, n Quantification of cells in the corpus callosum. Error bars represent SEM. b Olig1 (n = 7 sham, 10 LesVeh, and 11 LesTam). d Olig2 (n = 5 sham, 11 LesVeh, and 10 LesTam), f PDGFRα (n = 4 sham, 6 LesVeh, and 9 LesTam). g O4 (n = 9 sham, 7 LesVeh, and 6 LesTam). i CC1 (n = 5 sham and 5 LesTam). *p = 0.04, **p = 0.03. k GFAP (n = 5 sham, 5 LesVeh, and 7 LesTam). m Iba1 (n = 5 sham, 5 LesVeh, and 5 LesTam). n PDGFRa/GFP (green fluorescent protein) co-staining (n = 3 WT mice and 5 BMPR2 conditional knockout mice).

Article Snippet: The following antibodies were used: CNPase 1: 1,000 (Sternberger SMI91), GAPDH 1: 1,000 (Millipore MAB374), MBP 1: 1,000 (Sternberger SMI99), phosphorylated SMAD1/5/8 (pSMAD1/5/8) 1: 1,000 (Cell Signaling #9511), BMPR1a 1: 100 (Abgent AP2004a), BMPR1b 1: 1,000 (Abgent AP2005c), and BMPR2 1: 40 (Abgent AP2006a).

Techniques: Staining, Knock-Out

Journal: Developmental neuroscience

Article Title: Targeted Knockdown of Bone Morphogenetic Protein Signaling within Neural Progenitors Protects the Brain and Improves Motor Function following Postnatal Hypoxia-Ischemia

doi: 10.1159/000485379

Figure Lengend Snippet: Knockdown of bone morphogenic protein receptor 2 (BMPR2) following postnatal hypoxia-ischemia (HI) increases noncompacted myelin. Representative electron micrographs of the internal capsule 14 dpl in lesioned, vehicle-treated mice (LesVeh) (a) and lesioned, tamoxifen-treated mice (LesTam) (b). Quantification of myelin thickness (c), the G-ratio (ratio of the cross-sectional diameter of an axon to the diameter of that axon plus its surrounding myelin) (d), axon count (e), axon diameter (f), and noncompacted myelin (g). c–g n = 4 LesVeh and 4 LesTam. Error bars represent SEM. *p = 0.01.

Article Snippet: The following antibodies were used: CNPase 1: 1,000 (Sternberger SMI91), GAPDH 1: 1,000 (Millipore MAB374), MBP 1: 1,000 (Sternberger SMI99), phosphorylated SMAD1/5/8 (pSMAD1/5/8) 1: 1,000 (Cell Signaling #9511), BMPR1a 1: 100 (Abgent AP2004a), BMPR1b 1: 1,000 (Abgent AP2005c), and BMPR2 1: 40 (Abgent AP2006a).

Techniques: