r spondin1 antibody  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: Exogenous R-Spondin1 Induces Precocious Telogen-to-Anagen Transition in Mouse Hair Follicles

doi: 10.3390/ijms17040582

Figure Lengend Snippet: Exogenous R-spondin1 injection leads to precocious anagen entry. ( a ) Coomassie blue staining and immunoblotting of R-spondin1-Fc protein purified from the R-spondin1-293T cells; ( b ) TCF luciferase assay showing the bioactivity of the R-spondin1-Fc protein; ( c ) schedule for R-spondin1 injection assay; ( d ) pictures of mice injected with R-spondin1-Fc, Noggin or BSA (negative control) on PD80. Dotted circles show the injection site; ( e ) statistical analysis of the protein injection assay. Mice with precocious hair regrowth was regarded as + and the opposite as −; ( f ) X-gal staining in dorsal skin of TOP-Gal mice on PD55; ( g ) X-gal staining of dorsal skin of TOP-Gal mice injected with R-spondin1-Fc or BSA one week after the initial injection; an ( h ) X-gal staining of dorsal skin of TOP-Gal mice injected with R-spondin1-Fc or BSA about three weeks after the initial injection. CBB, Coomassie blue; R1-Fc, R-spondin1-Fc; PD, postnatal day; BSA, bovine serum albumin; Tel, telogen; Ana, anagen. Bar = 100 μm in ( f , g ). * p < 0.05.

Article Snippet: The level of R-spondin1-Fc protein was verified by CBB staining and immunoblotting with the R-spondin1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Injection, Staining, Western Blot, Purification, Luciferase, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Exogenous R-Spondin1 Induces Precocious Telogen-to-Anagen Transition in Mouse Hair Follicles

doi: 10.3390/ijms17040582

Figure Lengend Snippet: R-spondin1 activates Wnt/β-catenin signaling in bulge stem cells in vitro . ( a ) Morphology and green fluorescence of K14-H2B-GFP bulge stem cells in culture. The non-GFP cells were NIH-3T3 feeder cells; ( b ) RT-PCR showing the expression of Lgr4/5/6 in the cultured bulge stem cells (Bu). Mouse epidermal cells (Ep) were used as a positive control and H 2 O was used as a negative control (N); ( c ) Western blotting showing the activation of Wnt/β-catenin in bulge stem cells treated with LiCl, Wnt3a or/and R-spondin1. GAPDH was used as a loading control; ( d ) densitometric analysis of active β-catenin/β-catenin in c. Three independent experiments were performed for quantification; ( e – h ) qPCR results showing relative expression of Wnt/β-catenin target genes ( e ); cell cycle-related genes ( f ); genes associated with HFSC differentiation ( g ) and fate determination ( h ) in bulge stem cells treated with Wnt3a or/and R-spondin1. Bar = 100 μm. * p < 0.05; ** p < 0.01.

Article Snippet: The level of R-spondin1-Fc protein was verified by CBB staining and immunoblotting with the R-spondin1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: In Vitro, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Positive Control, Negative Control, Western Blot, Activation Assay, Control

Journal: PloS one

Article Title: Human RSPO1/R-spondin1 is expressed during early ovary development and augments β-catenin signaling.

doi: 10.1371/journal.pone.0016366

Figure Lengend Snippet: Figure 3. R-spondin1 weakly activates a b-catenin-responsive promoter. (A) The effect of WT RSPO1 on TCF-dependent transcriptional activation (left panel) was compared with that of a naturally-occurring RSPO1 mutant (MT) vector generating a protein lacking the first furin domain (right panel), using embryonic kidney tsa201 cells and the TOPFLASH reporter construct. Transient gene expression assays of increasing concentrations of plasmids encoding wild-type (WT) or mutant (MT) RSPO1 (0–100 ng/well) showed a mild activation (maximum 1.8-fold) of the TOPFLASH reporter with WT RSPO1 vector (ANOVA, p,0.001) and a complete lack of activity for the mutant. (B) Dose-dependent activation of the TOPFLASH reporter with increasing doses of beta-catenin (0–10 ng/well) (ANOVA, p,0.001). Luciferase data are reported as a mean 6 SEM of at least three triplicate experiments, standardized for Renilla co-expression (compared to basal value, **p,0.01; ***p,0.001). doi:10.1371/journal.pone.0016366.g003

Article Snippet: Fifty micrograms of protein extract from each sample was subjected to SDS/PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) and probed with the following antibodies: anti-HA probe (1:500) (sc-7392, Santa Cruz Biotechnology Inc.) or anti-R-spondin1 (1:300) (AF3474, R&D Systems).

Techniques: Activation Assay, Mutagenesis, Plasmid Preparation, Construct, Gene Expression, Activity Assay, Luciferase, Expressing

Journal: PloS one

Article Title: Human RSPO1/R-spondin1 is expressed during early ovary development and augments β-catenin signaling.

doi: 10.1371/journal.pone.0016366

Figure Lengend Snippet: Figure 4. R-spondin1 augments b-catenin signaling. (A) Co-transfection of increasing doses of wild-type (WT) RSPO1 (0–100 ng/well) with b- catenin (0–5 ng/well) showed dose-dependent augmentation of b-catenin signaling (left panel) (ANOVA: CTNNB1 0 ng, p,0.05; CTNNB1 2 ng, p,0.01; CTNNB1 5 ng, p = 0.07). A statistically significant synergistic effect was seen when doses of 10 ng RSPO1 and 50 ng RSPO1 were transfected on the background of increasing doses of CTNNB1 (ANOVA: RSPO1 10 ng, p,0.05; RSPO1 50 ng, p,0.05). No activity was seen after co-transfection of mutant (MT) RSPO1 (0–100 ng/well) (right panel). (B) Relative luciferase activity after stimulation of b-catenin transfected cells with Rspo1 peptide (0–3000 ng/ml) (ANOVA: CTNNB1 -, p,0.01; CTNNB1 +, p,0.001). Luciferase data are reported as a mean 6 SEM of at least three triplicate experiments, standardized for Renilla co-expression (*p,0.05; **p,0.01; ***p,0.001 compared to basal value without RSPO1 vector or peptide for that study). doi:10.1371/journal.pone.0016366.g004

Article Snippet: Fifty micrograms of protein extract from each sample was subjected to SDS/PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) and probed with the following antibodies: anti-HA probe (1:500) (sc-7392, Santa Cruz Biotechnology Inc.) or anti-R-spondin1 (1:300) (AF3474, R&D Systems).

Techniques: Cotransfection, Transfection, Activity Assay, Mutagenesis, Luciferase, Expressing, Plasmid Preparation

Journal: PloS one

Article Title: Human RSPO1/R-spondin1 is expressed during early ovary development and augments β-catenin signaling.

doi: 10.1371/journal.pone.0016366

Figure Lengend Snippet: Figure 5. Effect of DKK1 treatment on R-spondin1 augmentation of b-catenin signaling. (A) RSPO1/b-catenin co-transfected cells were treated 2 hours before transfection with different doses of DKK1 (0–400 ng/ml). After 24 h, cells were lysed and assayed for luciferase activity. No significant reduction in stimulation was seen (ANOVA: RSPO1 50 ng, p = 0.15). (B) Cells were treated with DKK1 (0–1000 ng/ml) and stimulated with Rspo1 peptide (0–2000 ng/ml). Luciferase activity was measured 24 h later. Luciferase data are reported as a mean 6 SEM of at least three triplicate experiments, standardized for Renilla co-expression. (N.S., not significant; *p,0.05). doi:10.1371/journal.pone.0016366.g005

Article Snippet: Fifty micrograms of protein extract from each sample was subjected to SDS/PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) and probed with the following antibodies: anti-HA probe (1:500) (sc-7392, Santa Cruz Biotechnology Inc.) or anti-R-spondin1 (1:300) (AF3474, R&D Systems).

Techniques: Transfection, Luciferase, Activity Assay, Expressing

Journal: PloS one

Article Title: Human RSPO1/R-spondin1 is expressed during early ovary development and augments β-catenin signaling.

doi: 10.1371/journal.pone.0016366

Figure Lengend Snippet: Figure 6. R-spondin1 can show nuclear and nucleolar localization. (A) Immunofluorescent microscopy was used to detect the cellular distribution of a WT pRSPO1-GFP vector in H295R cells (left panel). DAPI stained nuclei and merged images are shown (center and right panels, respectively) (magnification 20X). (B) A similar pattern of cellular distribution was obtained in a CHO cell line (left panel). Fluorescent labeling performed with an antibody against C23-nucleolar protein (red) revealed that the GFP-tagged WT RSPO1 protein shows strong nucleolar localization in some of these cells (center and right panels) (magnification 20X). (C) Immunofluorescent analysis of MT pRSPO1-GFP showed similar cellular distribution. (D) The strong nuclear localization was confirmed following Western blot analysis of nuclear and cytosolic extracts prepared from HEK293T cells that had been transfected with either WT or MT pRSPO1-HA constructs. doi:10.1371/journal.pone.0016366.g006

Article Snippet: Fifty micrograms of protein extract from each sample was subjected to SDS/PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) and probed with the following antibodies: anti-HA probe (1:500) (sc-7392, Santa Cruz Biotechnology Inc.) or anti-R-spondin1 (1:300) (AF3474, R&D Systems).

Techniques: Microscopy, Plasmid Preparation, Staining, Labeling, Western Blot, Transfection, Construct