Fig. 7: Head and middle region of Lnc-ip65 binds to p65 hinders its phosphorylation.
From: Disparate macrophage responses are linked to infection outcome of Hantan virus in humans or rodents
a, b RIP experiments assessing the interaction between indicated lncRNAs and crucial TFs. Samples are acquired from the mBMDM exogenously expressing the indicated proteins and lncRNAs (a, n = 4) or in HTNV-infected mBMDM from 0 hpi to 48 hpi (b, n = 4). c Representative immunofluorescent analysis of three independent experiments for lnc-ip65 with different probes (FISH, probe 1 targeted to the 1–1000 nt, and probe 2 to 1001-2000 nt) and GFP-65 in RAW264.7 cells at 36 hpi (MOI = 1). Scale bars, 50 μm. d Representative immunoblot analysis of three independent experiments for protein phosphorylation in WT and lnc-ip65−/− mBMDM (MOI = 5). e Representative live cell imaging of three independent experiments depicting the translocation of GFP-p65 in WT and lnc-ip65−/− mBMDM (MOI = 1). Live cell imaging is recorded from 24 to 36 hpi. Scale bars, 10 μm. f Immunoblot confirmation for truncated p65 expression (upper) and RIP analysis for the interaction of truncated p65 with lnc-ip65 in RAW264.7 cells (bottom). g Representative immunofluorescent analysis of three independent experiments for RAW264.7 cells overexpressing p65 mutants and lnc-ip65. The co-localization of truncated p65 and lnc-ip65 is shown with overexposure. Scale bars, 10 μm. h Representative immunoblot analysis of three independent experiments for p65 and IκBα in RAW264.7 cells that were exogenously expressed with different lnc-ip65 mutants at 24 hpi with an MOI of 5. i Representative immunofluorescent analysis of three independent experiments for the subcellular localization of myc-p65 and lnc-ip65 mutants in HTNV-infected RAW264.7 cells (MOI = 5, 24 hpi). Scale bars, 10 μm. j qRT-PCR analysis for TNFα mRNA expression from (h). Vector as control (vs. 1–3514, 1–1000, 1–500, 501–1000, 1001–2000, 1001–1500 or 1501–2000), p < 0.0001/ = 0.0024/ = 0.0004/ = 0.0028/<0.0001/ < 0.0001/<0.0001. k RIP analysis for the interaction of truncated p65 with lnc-ip65 mutants in RAW264.7 cells. Data are shown as the mean ± SEM, and are representative of three independent experiments. Analysis is performed using one-way ANOVA (Dunnett’s multiple comparisons test). *p < 0.05, **p < 0.01, ***p < 0.001. Molecular weight markers are shown to the left of the blots in kDa, and antibodies used are indicated to the right. Source data are provided as a Source Data file.
