lps plus atp stimulation  (InvivoGen)

Journal: PLOS Biology

Article Title: Noncanonical contribution of microglial transcription factor NR4A1 to post-stroke recovery through TNF mRNA destabilization

doi: 10.1371/journal.pbio.3002199

Figure Lengend Snippet: (A) Immunoblot analysis of NR4A1 in WT and Nr4a1 -/- primary microglia treated with or without ATP (1 mM) + LPS (100 ng/ml). (B) Immunoblot analysis of NR4A1 expression in the cytoplasmic and nuclear fractions of untreated and ATP+LPS-treated primary microglia. (C) Co-IP-MS/MS analysis of Flag-NR4A1-interacting proteins. (D, E) Representative GO terms of cellular components and molecular functions enriched in Flag-NR4A1-interacting proteins. (F) Representative images of untreated and ATP+LPS-treated primary microglia showing colocalization of NR4A1 with DCP1A. Scale bar, 50 and 5 μm. (G, H) In situ proximity ligation assay assessing the interactions between NR4A1 and DCP1A (red) in negative control, unstimulated and ATP+LPS-treated primary microglia. Scale bar, 50 and 10 μm ( n = 50 cells counted in each group). (I) Immunoblot analysis of DCP1A in BV2 cell (expressing FLAG-NR4A1) lysates immunoprecipitated with an anti-FLAG antibody. Data are presented as mean ± SEM. In ( H ), one-way ANOVA with post hoc Dunnett’s test. *** P < 0.001. The underlying data for this figure can be found in . The original blot for this figure can be found in . Co-IP, coimmunoprecipitation; WT, wild-type.

Article Snippet: NR4A1-binding RNAs were precipitated from 5.0 × 10 7 ATP+LPS-stimulated BV2 cells with anti-NR4A1 (sc-166166, Santa Cruz Biotechnology) antibody, and rRNAs were then removed using Ribo-off rRNA Depletion Kit (N406-01/02, Vazyme) according to the manufacturer’s instruction.

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay, Tandem Mass Spectroscopy, In Situ, Proximity Ligation Assay, Negative Control, Immunoprecipitation

Journal: PLOS Biology

Article Title: Noncanonical contribution of microglial transcription factor NR4A1 to post-stroke recovery through TNF mRNA destabilization

doi: 10.1371/journal.pbio.3002199

Figure Lengend Snippet: (A) RIP analysis of NR4A1-bound Tnf mRNA in untreated and ATP+LPS-treated BV2 cells ( n = 3 biological repeats in each group). (B) RNA stability assay of Tnf mRNA in ATP+LPS-activated WT and Nr4a1 -/- primary microglia at the indicated time points after actinomycin D treatment ( n = 4 biological repeats in each group). (C, D) mRNA ( C ) ( n = 3 biological repeats in each group) and protein ( D ) levels of Nr4a1 in unstimulated and ATP+LPS-activated primary microglia treated with DMSO or TSA (50 nM). (E) Tnf mRNA levels in unstimulated and ATP+LPS-treated primary microglia treated with DMSO or TSA (50 nM) ( n = 3 biological repeats in each group). (F) Relative reduction in Tnf expression induced by TSA treatment in ATP+LPS-treated WT and Nr4a1 -/- primary microglia (calculated from E ) ( n = 3 biological repeats in each group). (G, H) RNA stability assay of Tnf mRNA in ATP+LPS-treated WT and Nr4a1 -/- primary microglia treated with TSA at the indicated time points after actinomycin D treatment ( n = 3 biological repeats in each group). Data are presented as mean ± SEM. In ( A ), ( C ), ( E ), two-way ANOVA with post hoc Bonferroni’s test. In ( B ), ( G ), ( H ), two-way ANOVA. In ( F ), two-tailed unpaired Student’s t test. * P < 0.05; ** P < 0.01; *** P < 0.001. The underlying data for this figure can be found in . The original blot for this figure can be found in . RBP, RNA-binding protein; TSA, trichostatin A; WT, wild-type.

Article Snippet: NR4A1-binding RNAs were precipitated from 5.0 × 10 7 ATP+LPS-stimulated BV2 cells with anti-NR4A1 (sc-166166, Santa Cruz Biotechnology) antibody, and rRNAs were then removed using Ribo-off rRNA Depletion Kit (N406-01/02, Vazyme) according to the manufacturer’s instruction.

Techniques: Stability Assay, Expressing, Two Tailed Test, RNA Binding Assay

Journal: PLOS Biology

Article Title: Noncanonical contribution of microglial transcription factor NR4A1 to post-stroke recovery through TNF mRNA destabilization

doi: 10.1371/journal.pbio.3002199

Figure Lengend Snippet: (A) Binding motif identified by HOMER with NR4A1-binding peaks. (B) The distribution (left) and enrichment (right) of NR4A1-binding peaks identified by RIP-seq. (C, D) Immunoblot analysis of NR4A1 in the cytoplasmic fraction of ATP+LPS-treated primary microglia pulled down by m 6 A-containing or unmethylated oligonucleotides. (E) SRAMP software prediction of the m 6 A sites in Tnf mRNA. (F) m 6 A-RIP-qPCR analysis of m 6 A enrichment in the CDS and 3′ UTR of Tnf mRNA in BV2 cells ( n = 3 biological repeats in each group). (G) Luciferase activities of the 5′ UTR, CDS, and 3′ UTR of Tnf in HEK293T cells overexpressing NR4A1 or empty vector ( n = 3 biological repeats in each group). (H) m 6 A site mutations in the CDS and 3′ UTR of Tnf mRNA. (I, J) Relative luciferase activities of WT or mutant CDS of Tnf ( I ) and WT or mutant 3′ UTR of Tnf ( J ) in HEK293T cells overexpressing NR4A1 (normalized to Fluc/Rluc in HEK293T cells with empty vector) ( n = 3 biological repeats in each group). Data are presented as mean ± SEM. In ( F ), ( G ), ( I ), two-tailed unpaired Student’s t test. In ( J ), one-way ANOVA with post hoc Dunnett’s test. * P < 0.05; ** P < 0.01; *** P < 0.001. The underlying data for this figure can be found in . The original blot for this figure can be found in . RIP-seq, RNA-binding protein immunoprecipitation sequencing; WT, wild-type.

Article Snippet: NR4A1-binding RNAs were precipitated from 5.0 × 10 7 ATP+LPS-stimulated BV2 cells with anti-NR4A1 (sc-166166, Santa Cruz Biotechnology) antibody, and rRNAs were then removed using Ribo-off rRNA Depletion Kit (N406-01/02, Vazyme) according to the manufacturer’s instruction.

Techniques: Binding Assay, Western Blot, Software, Luciferase, Plasmid Preparation, Mutagenesis, Two Tailed Test, RNA Binding Assay, Immunoprecipitation, Sequencing