Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: Dynamic expression of Rbp4 in mouse ovaries during the different stages of development and the estrous cycle. (A) Real-time qPCR analyses of Rbp4 mRNA levels in mouse ovaries during different developmental stages. Data are presented as means ± SEM, n = 8. (B) Relative Rbp4 mRNA levels in mouse ovaries during diestrus (D), proestrus (P), estrus (E), and metestrus (M). Data are presented as mean ± SEM, n = 8. RBP4 protein levels in the ovaries of mice during the estrous cycle were determined via Western blot analyses (D), and blots were quantified via densitometry (C). The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. Means with different letters represent a significant difference at the 0.05 level

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Expressing, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: Effects of gonadotropins and ovarian steroids on the expression of Rbp4 in the 3-week-old mouse ovary. (A) Rbp4 mRNA expression levels in the ovaries of mice treated with FSH, LH, or FSH + LH for 48 h. Data are presented as means ± SEM, n = 8. (B) Rbp4 mRNA expression levels in the ovaries of mice treated with 17β-estradiol for 24 or 48 h. Data are presented as mean ± SEM, n = 8. RBP4 protein levels in the ovaries of mice treated with FSH or LH were determined using Western blot analyses (D), and blots were quantified via densitometry (C). The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. Means with different letters represent a significant difference at the 0.05 level

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Expressing, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: Localization of RBP4 protein in the ovaries of the immature mice (3 weeks of age) treated with or without FSH for 48 h. Negative controls (NC), which were incubated with 5% goat serum (NC1) or PBS (NC2) instead of the primary antibody, were used to validate the specificity of RBP4 antibody. Positive signals for RBP4 were detected in granulosa and theca cell layers. Bar = 40 μm

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Incubation

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: Effects of gonadotropins on the expression of Rbp4 in mouse granulosa cells. (A) Relative Rbp4 mRNA levels in mouse granulosa cells treated with FSH and LH for 24 h. Data are presented as means ± SEM, n = 4. RBP4 protein levels in mouse granulosa cells treated with FSH and LH for 24 h were determined using Western blot analyses (C), and blots were quantified via densitometry (B). The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. Means with different letters represent a significant difference at the 0.05 level

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Expressing, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: PKA mediates the FSH induction of Rbp4 in mouse granulosa cells. (A) Activation of PKA by cAMP and FSH increased Rbp4 mRNA levels. Data are presented as means ± SEM, n = 4. (B) The PKA inhibitor H-89 abrogated FSH induced Rbp4 mRNA expression. Granulosa cells were pre-incubated with 10 μM H-89 for 1 h, followed by incubation with 100 IU/L FSH for 24 h. Data are presented as means ± SEM, n = 4. (C) Densitometric quantification of Western blots for RBP4 (D). The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. Means with different letters represent a significant difference at the 0.05 level

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Activation Assay, Expressing, Incubation, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: HMGA1, SF-1, and LRH-1 are involved in the FSH induction of Rbp4 in mouse granulosa cells. mRNA (A) and protein (D) levels of Hmga1 in mouse granulosa cells transfected with 50 nM Hmga1 siRNA. The mRNA (B) and protein (E) levels of Sf-1 in mouse granulosa cells transfected with 50 nM Sf-1 siRNA. The mRNA (C) and protein (F) levels of Lrh-1 in mouse granulosa cells transfected with 50 nM Lrh-1 siRNA. The mRNA levels were determined using real-time PCR. Data are presented as means ± SEM, n = 4. The protein levels were determined using Western blot analyses, and blots were quantified via densitometry. The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. *p < 0.05, ** p < 0.01. (G) The mRNA levels of Rbp4 in mouse granulosa cells transfected with NC siRNA, Hmga1 siRNA, Sf-1 siRNA, or Lrh-1 siRNA (50 nM). Data are presented as means ± SEM, n = 4. (H), (I), and (J) The RBP4 protein levels in mouse granulosa cells transfected with NC siRNA, Hmga1 siRNA, Sf-1 siRNA, or Lrh-1 siRNA (50 nM). The protein levels were determined using Western blot analyses and blots were quantified via densitometry. The protein levels were normalized to GAPDH. Data are presented as means ± SEM, n = 3. Means with different letters represent a significant difference at the 0.05 level

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Western Blot

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Regulation by FSH of the dynamic expression of retinol-binding protein 4 in the mouse ovary

doi: 10.1186/s12958-018-0348-8

Figure Lengend Snippet: Schematic overview of the proposed pathway involved in the induction of Rbp4 by FSH. FSH binds to its receptor (FHR) and elevates cAMP levels, which activates PKA. PKA then stimulates the expression of HMGA1, SF-1, and LRH-1, and promotes Rbp4 transcription

Article Snippet: Sections were incubated overnight at 4 °C with mouse monoclonal antibody specific to RBP4 at a 1:200 dilution in PBS (Proteintech, USA).

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Gastric cancer-derived LBP promotes liver metastasis by driving intrahepatic fibrotic pre-metastatic niche formation

doi: 10.1186/s13046-023-02833-8

Figure Lengend Snippet: LBP secretion is associated with GC metastasis to liver and correlated with worse prognosis. A Heatmap of 49 dysregulated secreted proteins detected by DIA-MS secretomic analysis of serum samples from five patients with GC-LM and five patients with GC-NLM. B Clustered heatmap analysis of the top 200 dysregulated mRNAs of 24 pairs of GC tissues and matched normal gastric tissues (|foldchange|> 2; p < 0.05). C Serological levels of LBP and RBP4 were detected by ELISA in the serum of 30 patients with GC-LM, 30 patients with stage II/III GC and 20 healthy volunteers. D WB verified the expression levels of LBP in GC tissues and matched adjacent normal gastric tissues. E Representative IHC images of LBP in paired primary tumours and LM from the same GC patients. Scale bar, 100 μm. F Comparisons of LBP expression in primary GC tumors and LM based on IHC results. G ROC curve analysis was applied based on the previous serological LBP levels of 30 GC-LM patients and 30 GC-NLM patients. H Liver metastasis-free survival analysis of an additional cohort of GC patients based on serological LBP levels. The cut-off value was determined according to the ROC curve. Data are representative of three independent experiments. Data are shown as mean ± SEM, and p values were determined by one-way ANOVA test ( C ) or log rank test ( H ) (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: ELISA kits were used to determine the levels of human RBP4 (KE00056, Proteintech, China), human LBP (KE00134, Proteintech, China), human TGF-β1 (BMS249-4, Thermo Fisher Scientific, USA) in serum samples or cell culture supernatants, following the manufacturer’s instruction.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Nature Communications

Article Title: Characterising the RNA-binding protein atlas of the mammalian brain uncovers RBM5 misregulation in mouse models of Huntington’s disease

doi: 10.1038/s41467-023-39936-x

Figure Lengend Snippet: a Experimental workflow of brain-pCLAP: Brain halves from four mice (4X) were homogenized and the right half was cross-linked using UV-light (254 nm). Cells were spun down, and lysates were LysC treated, pre-cleared with empty beads and RNA-peptide complexes were purified using oligo(dT) beads. After elution and trypsin treatment, peptides were analysed using LC-MS/MS. The same experiment was conducted in parallel without the pre-clearance step. b PCA plot of MS data from all samples. c ‘Filter1’: Volcano plot comparing peptide intensities between cross-linked (XL) and non-cross-linked (noXL) samples (two-tailed t test with multiple-testing correction). Each detected peptide is represented by a dot and dots highlighted in black represent peptides from proteins with canonical RBDs. The lines represent thresholds for significant enrichment in the cross-linked (XL) and non-cross-linked samples (noXL). d Percentage of proteins previously annotated as RNA-binding in the proteome and proteins inferred from peptides identified by pCLAP before and after the two data filtering steps. e Number of proteins with shown canonical RBDs in the proteome and in brain-pCLAP with and without the data filtering steps. The p value indicates the enrichment of the fraction of proteins containing each domain in the final brain-pCLAP dataset over the fraction of these protein in the brain proteome (one-tailed Fischer’s exact test with FDR correction) (colours as in ( d )). f Abundance distribution of total brain proteome and brain-pCLAP. The middle of the box signifies the mean, the box boundaries represent the 25th and 75th percentile and the whiskers reach to the minimum and maximum value of the dataset. n = 4 biologically independent animals. p value calculated using two-tailed t test, **** p < 0.0001. Source data are provided as a Source data file. g Donut plot showing the number of proteins identified by brain-pCLAP, which have also been identified as RBPs from cell line based studies and the number of known RBPs among the proteins specific to this study. h GO term enrichment for cellular compartment for proteins identified as RBPs specifically by brain-pCLAP or those overlapping with cell line based studies. i String network of the 95 proteins identified in this study as RNA binding not identified in previous cell culture-based studies.

Article Snippet: All vectors expressing GFP-tagged candidate RBPs were ordered from GeneScript.

Techniques: Purification, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, RNA Binding Assay, One-tailed Test, Cell Culture

Journal: Nature Communications

Article Title: Characterising the RNA-binding protein atlas of the mammalian brain uncovers RBM5 misregulation in mouse models of Huntington’s disease

doi: 10.1038/s41467-023-39936-x

Figure Lengend Snippet: a Locations of RNA-binding peptides identified by brain-pCLAP mapped to linear depictions of RBPs. Tissue expression data taken from brain atlas . b Schematic of membrane bound synaptic vesicle proteins identified as RNA-binding by brain-pCLAP, showing their trans-membrane domains as well as the locations of peptides identified by brain-pCLAP as RNA-binding regions (blue stars). c Data demonstrating RNA-binding ability of PGRMC1 and PGRMC2. Left: Volcano plots (as in Fig. ) with peptides from PGRMC1 or PGRMC2 marked in black. Middle: Schematic depiction of both proteins showing their trans-membrane and cytochrome b5-like domains and where the peptides identified by brain-pCLAP are located. Right: CLIP autoradiography for GFP-tagged PGRMC1 and PGRMC2, with visualization of the isotope labelled protein-RNA complex. The left lane is a no cross-link control, followed by the cross-linked samples from highest to lowest concentration of RNase (dilutions 1:10, 1:100 or 1:1000). The arrow points to the expected size of the protein. Each lane of each CLIP experiment represents a replicate of independently grown cells.

Article Snippet: All vectors expressing GFP-tagged candidate RBPs were ordered from GeneScript.

Techniques: RNA Binding Assay, Expressing, Membrane, Autoradiography, Control, Concentration Assay