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: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: TIPS construction and validation. ( a ): Based on 302 TIDEGs, the training set is screened using univariate Cox analysis to identify 18 prognosis-related genes. Unifactorial analysis of TIDEGs in the training set. ( b ): Three TICPGs are identified using multivariate Cox analysis, including KLRK1 (β=-0.303), LTB (β=-0.562), and RBP4 (β = 0.161). The risk score formula is as follows: risk score=(-0.303×KLRK1)+(-0.562×LTB)+(0.161×RBP4). ( c ): Survival curve analysis of TIPS in the high-risk group vs. low-risk group in the training set, ROC curves, distribution of risk scores, distribution of survival statuses, and heatmap of TICPGs. The area under the curve (AUCs) of TIPS at 1, 3, and 5 years are 0.7, 0.704, and 0.739, respectively. ( d ): Heatmap of TIPS in the test set of high-risk group vs. low-risk group survival curve analysis, ROC curve, risk score distribution, survival status distribution, TICPGs heat map. In the test and full sets ( n = 158), the high- and low-risk groups are divided based on optimal cutoff value; KM curves showed that the prognosis of the low-risk group was significantly better ( p < 0.05). ROC analysis shows the AUCs in the test set at 1, 3, and 5 years of 0.737, 0.64, and 0.647, respectively. ( e ): TIPS in the full set of high-risk group vs. low-risk group survival curve analysis, ROC curve, risk score distribution, survival status distribution, TICPGs heat map. Corresponding AUCs in the full set are 0.713, 0.677, and 0.711, respectively. ( f ): Full set of univariate analysis of TIPS vs. traditional clinical indicators. ( g - i ): ROC curve of the AUC values in TIPS at 1, 3, and 5 years. (j): Full set of multifactorial analyses of TIPS versus STAGE, T versus N staging. DEGs, differentially expressed genes; EMT, epithelial-mesenchymal transition; ROC, receiver operating characteristic; TIPS, TME immune-related genes prognostic signature; TIDEGs, TME immune-related DEGs; TME, tumor microenvironment.

Article Snippet: Transcriptome sequencing was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

Techniques: Biomarker Discovery

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: Effects of RBP4 overexpression or knockdown on TSCC cell proliferation, migration, and invasion. ( a ): Differential analysis of KLRK1, LTB, and RBP4 expression in tumor tissues vs. paired paracancerous normal tissues in the TCGA-TSCC dataset. ( b ): In the GSE13601 ( p = 5e-06), GSE78060 ( p = 0.082), and GSE31056 ( p = 1e-05) datasets, the RBP4 expression in tumor samples vs. paired paracancerous normal tissue samples. RBP4 differential expression analysis in tumor samples relative to paraneoplastic normal tissue samples in GSE13601 , GSE78060 , and GSE31056 datasets. The left panel shows the volcano plots of DEGs; the right panel shows the box plots of RBP4 expression levels in tumor samples versus paraneoplastic normal samples. ( c ): Immunohistochemistry observation of RBP4 expression in TSCC and paraneoplastic tissues. The upper panel shows the expression of RBP4 in TSCC tissues. Scale bar: 200 μm. The lower panel shows the expression of RBP4 in paraneoplastic tissues. Scale bar: 50 μm. ( d - g ): Overexpression or knockdown of RBP4 in CAL27 and SCC-15 cells using lentivirus and validated by qRT-PCR with western blotting experiments. ( h ): CCK-8 method is used to analyze the effect of changes in RBP4 levels on the proliferative ability of TSCC cells. ( i ): In vivo experiments validate the effect of changes in RBP4 expression levels on the TSCC cell proliferation rate. ( j ): Effects of changes in RBP4 expression levels on TSCC cell migration ability were observed by scratch assay. ( k ): Effects of changes in RBP4 expression levels on TSCC migration invasion ability were observed by Transwell assay. DEGs, differentially expressed genes; TCGA, The Cancer Genome Atlas Program; TSCC, tongue squamous cell carcinoma. (*Compared with NC, * p < 0.05, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

Techniques: Over Expression, Knockdown, Migration, Expressing, Quantitative Proteomics, Immunohistochemistry, Quantitative RT-PCR, Western Blot, CCK-8 Assay, In Vivo, Wound Healing Assay, Transwell Assay

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: Mechanism study of RBP4 affecting TSCC cell proliferation, migration, and invasion. ( a ): Bioinformatic analysis of the effect of RBP4 on the PI3K/Akt/mTOR signaling pathway. ( b ): Western blotting to detect the effect of changes in the expression level of RBP4 on the activation level of the PI3K/Akt/mTOR pathway in TSCC cells. ( c ): After addition of 740 Y-P (0.2 mM), western blotting detects the alteration of the effect of RBP4 on the PI3K/Akt/mTOR pathway. ( d ): CCK-8 method is used to analyze the changes in the effect of RBP4 on the proliferative capacity of TSCC cells after the addition of 740 Y-P (0.2 mM). ( e - f ): To explore the mechanism by which changes in RBP4 levels affect TSCC cell migration and invasion, we determine the changes in Snail expression in RBP4 overexpressing and RBP4 knockdown TSCC cells. qRT-PCR and western blotting are used to detect the effect of RBP4 on the expression level of Snail and EMT-related proteins. ( g ): PI3K agonist treatment reverses the effects of RBP4 overexpression on Snail/E-cadherin/N-cadherin/Vimentin regulation. Western blotting to detect the changes of the effect of RBP4 on the PI3K/Akt/mTOR pathway after the addition of 740 Y-P (0.2 mM). ( h ): PI3K agonist treatment enhances cell migration and invasion, suggesting that RBP4 suppresses the TSCC malignant phenotype by inhibiting the PI3K/Akt/mTOR-Snail axis. Scratch assay to detect the changes of the effect of RBP4 on the migration ability of TSCC cells after the addition of 740 Y-P (0.2 mM). Changes in the effect of TSCC cell migration ability. ( i ): Transwell assay to detect changes in the effect of RBP4 on TSCC cell invasion ability after addition of 740 Y-P (0.2 mM). TSCC, tongue squamous cell carcinoma. (*Compared with NC, * p < 0.05, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

Techniques: Migration, Western Blot, Expressing, Activation Assay, CCK-8 Assay, Knockdown, Quantitative RT-PCR, Over Expression, Wound Healing Assay, Transwell Assay

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: RBP4 in TSCC cells affects macrophage phenotypic transformation. To investigate the association between RBP4 expression levels and tumor-infiltrating macrophage phenotypes in TSCC tissues, we perform a correlation bioconfidence analysis using the GEO database. ( a ): In the GSE13601 dataset, the left graph represents the distribution of macrophage M1 polarization in the subgroup of tumor samples versus paracancerous samples, and the right graph represents the distribution of macrophage M1 polarization in the subgroup of samples with high and low RBP4 expression in tumor tissues. ( b ): In the GSE31056 dataset, the left graph represents the distribution of macrophage M1 polarization in the subgroup of tumor samples versus paracancerous samples, and the right panel represents the distribution of macrophage M1 polarization in the subgroup of samples with high and low tumor tissue RBP4 expression. Significance is calculated using the Wilcoxon rank-sum test. ( c ): CD86 and RBP4 expression levels are determined using immunohistochemical staining of paraneoplastic and TSCC tissues. ( d ): Flow cytometry is performed to detect phenotypic changes in macrophages co-cultured with TSCC. TSCC, tongue squamous cell carcinoma. (*Compared with NC, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

Techniques: Transformation Assay, Expressing, Immunohistochemical staining, Staining, Flow Cytometry, Cell Culture

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: RBP4 promotes macrophage M1 polarization by activating the TLR4/NF-κB pathway. ( a ): KEGG Bar Charts of human recombinant RBP4 protein-treated M0 macrophage cells after transcriptome sequencing. ( b ): Correlation analysis of RBP4 and TLR4 protein expression using Spearman and Pearson methods. ( c ): Western blotting assay to detect the effect of human recombinant RBP4 on macrophage TLR4/NF-KB pathway and the change of the effect of RBP4 action after the addition of TAK242. ( d , e ): qRT-PCR and western blotting are used to detect the effect of the human recombinant RBP4 protein on the macrophage phenotype and the effect of RBP4 action after the addition of TAK242. ( f , g ): Western blotting experiments are used to detect the effects of changes in RBP4 expression levels on the macrophage TLR4/NF-κB pathway in a Transwell co-culture system. ( h ): Western blotting experiments are used to detect the effects of tumor-secreted RBP4 on the macrophage TLR4/NF-κB pathway after the addition of TAK242. Changes in activation level. ( i ): Western blotting experiments are used to detect the effect of tumor-secreted RBP4 on macrophage phenotype after addition of TAK242 in Transwell co-culture system. TAK242 treatment blocks RBP4 overexpression induced by TSCC cells in the macrophage TLR4/p-NF-κB upregulation and M1-type polarization. KEGG, Kyoto Encyclopedia of Genes and Genomes; TSCC, tongue squamous cell carcinoma. (*Compared with Control, * p < 0.05, ** p < 0.01, *** p < 0.001,*** p < 0.0001).

Article Snippet: Transcriptome sequencing was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

Techniques: Recombinant, Sequencing, Expressing, Western Blot, Quantitative RT-PCR, Co-Culture Assay, Activation Assay, Over Expression, Control

Journal: Scientific Reports

Article Title: RBP4 interferes with tongue squamous cell carcinoma progression by inhibiting the PI3K/AKT signaling pathway and promoting macrophage M1-type polarization

doi: 10.1038/s41598-026-39915-4

Figure Lengend Snippet: Effect of RBP4 on macrophage phenotype. ( a ): Animals were grouped according to the injection of different cells, and the tumor volume, growth curve, and mass were measured and recorded. ( b ): The levels of CD86 and CD206 cell infiltration in the sections of transplanted tumors from each group are analyzed using immunofluorescence staining. ( c ): Changes in the activation level of the TLR4/NF-KB pathway in each group of transplanted tumor tissues are detected using Western blotting. ( d ): Mechanistic diagram of the effects of changes in RBP4 levels on the proliferation, migration, and invasion abilities of TSCC cells and the effects of tumor-secreted RBP4 on the phenotype of TAMs. TAMs, tumor-associated macrophages; TSCC, tongue squamous cell carcinoma. (*Compared with RAW264.7 + Control, * p < 0.05, ** p < 0.01).

Article Snippet: Transcriptome sequencing was performed by Genechem, based on the Illumina sequencing platform for transcriptome sequencing (RNA-Seq) of a recombinant human RBP4 (2 μg/ml, HY- P70471 , MCE) protein-treated M0 macrophage cell model. A KEGG pathway enrichment analysis was performed using the R software “clusterProfiler” package after obtaining the differential genes based on gene expression analysis.

Techniques: Injection, Immunofluorescence, Staining, Activation Assay, Western Blot, Migration, Control

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