human sema7a ab  (R&D Systems)

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Primers used.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques:

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Overview of regulated genes and their known relevant function.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Microarray, Expressing, Migration

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Regulation of NGC expression by QKI. (a) Heat map of expression of NGCs in monocytes and macrophages determined by RNA-seq from QKI patient and sibling in log2 scale (Lighter blue indicates higher expression; n = 1). (b) Fold change of NGC expression comparing QKI patient’s monocyte and macrophage to her sibling’s. (c) In macrophages treated with GapmeR antisense oligonucleotides, there is a significant positive correlation between QKI and SEMA7A expression measured by quantitative PCR analysis. Gene expression is expressed as copies per GAPDH. QKI: quaking.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Visualisation of genomic annotations in 3′UTR region of SEMA7A gene. The region in 3′UTR of SEMA7A in the human genome (hg19 Chromosome 15 q24.1, bp 74,701,500–74,703,000) is annotated with aspects of miRNA binding sites, QKI targeting sites (experimental evidence based), QRE ( in silico alignment of ‘ACUAA motif’) and SEMA7A exon. The microRNA binding information was extracted from ‘miRbase’ and was filtered by expression in monocytes/macrophages and miRNA interaction score. Interactions between RNA binding proteins (RBPs) to genomic DNA were obtained from technique duplicates of eCLIP-seq in the myelogenous K562 cells.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Binding Assay, In Silico, Expressing, RNA Binding Assay

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: RNA-immunoprecipitation showing quaking protein binding to the 3′UTR of SEMA7A. (a) RNA-immunoprecipitation in Hek293T cells overexpression QKI5 using an IgG control or QKI5 Ab. QKI5 and GAPDH mRNA abundance in immune-precipitated fraction was determined by qPCR. Results are presented relative to IgG immunoprecipitation. Data are the mean ± SEM; n = 5; * P < 0.05. (b) RNA-immunoprecipitation in Hek293T cells overexpression QKI5 and 3′UTR of SEMA7A using an IgG control or QKI5 Ab. SEMA7A 3’UTR and GAPDH mRNA abundance in immune-precipitated fraction was determined by qPCR. Results are presented relative to IgG immunoprecipitation. Data are the mean ± SEM; n = 3; * P < 0.05.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: RNA Immunoprecipitation, Protein Binding, Over Expression, Control, Immunoprecipitation

Journal: Innate Immunity

Article Title: Comprehensive analysis of neuronal guidance cue expression regulation during monocyte-to-macrophage differentiation reveals post-transcriptional regulation of semaphorin7A by the RNA-binding protein quaking

doi: 10.1177/1753425920966645

Figure Lengend Snippet: Role for SEMA7A in monocyte differentiation into pro-inflammatory macrophages. (a) Bright field microscope images from THP-I monocytes and macrophages. (b) Representative immunoblot analysis of SEMA7A or ACTB (loading control) in protein lysates of THP-I monocytes and macrophages. (c) Bright field microscope images from THP-I macrophages transduced with anti-SEMA7A shRNA (sh-SEMA7A) or scrambled shRNA (sh-Ctrl). Elongated phenotype indicated by #. (d) Ratio of TNF-α and IL-10 mRNA expression of sh-SEMA7A or sh-Ctrl THP-I macrophages. Data are the mean ± SEM; n = 4; * P < 0.05.

Article Snippet: Membranes were incubated with human SEMA7A Ab (1:10,000, AF 2068; R&D systems) or GAPDH Ab (1:1000, 5174; Cell Signaling Technology) in blocking buffer overnight at 4°C.

Techniques: Microscopy, Western Blot, Control, Transduction, shRNA, Expressing