Journal: Food Science & Nutrition

Article Title: Gastrodia elata Polysaccharide Attenuates Aging by Modulating Oxidative Stress, Inflammation, and Gut Microbiota

doi: 10.1002/fsn3.72413

Figure Lengend Snippet: GEP alleviates inflammation in aging mice by inhibiting the TLR4/NF‐κB signaling pathway. (A–C) Levels of IL‐1β (A), IL‐6 (B), and TNF‐α (C) in mouse serum. (D–F) Levels of IL‐1β (D), IL‐6 (E), and TNF‐α (F) in mouse brain tissues. (G, H) Expression levels of representative proteins (TLR4 and P‐P65) of the TLR4/NF‐κB signaling pathway in mouse brain tissues (G) and their quantitative analysis (H, I). All experimental data are presented as mean ± SD. n = 6, ns > 0.05, #### p < 0.0001 versus Control group; ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus Aging group.

Article Snippet: Membranes were incubated with primary antibodies against Nrf2 (1:400, sc‐365949, Santa Cruz), Keap1 (1:800, A25951 , ABclonal), HO‐1 (1:500, ab13248, Abcam), NQO1 (1:5000, A19586, ABclonal), TLR4 (1:600, Sc‐293072; Santa Cruz), P65 (1:600, Sc‐8008; Santa Cruz), phospho‐P65 (1:600, Sc‐136548, Santa Cruz), and GAPDH (1:10,000, 60004‐1‐Ig, Proteintech).

Techniques: Expressing, Analysis, Control

Journal: Life Science Alliance

Article Title: The noncoding RNA GRAS1 interacts with NKAP and protects against DNA damage and cell death

doi: 10.26508/lsa.202403105

Figure Lengend Snippet: (A) RNA antisense purification–mass spectrometry experimental outline. (B) RT-qPCR analysis of GRAS1, U1 snRNA, and GAPDH mRNA in nuclear and cytoplasmic fractionations of A549 cells (left panel). Immunoblot of Lamin A/C as a marker for nuclear fraction, and GAPDH as a marker for cytoplasmic fraction (right panel); N = 3. (C) Validation of GRAS1 RNA capture and recovery by biotinylated antisense oligonucleotide probes against GRAS1. RT-qPCR analysis of GRAS1 RNA and TUBB control mRNA in three fractions, including input with probes added, unbound sample flowthrough, and elution; N = 3. (D) Total proteins identified in RAP-MS of GRAS1 RNA, U1 snRNA positive control, and luciferase mRNA negative control elution samples. (E) GRAS1 interactome proteins identified by RAP-MS, with at least two unique peptides, after excluding proteins that were also found in U1 and luciferase control captures (left panel). The top 10 GRAS1-interacting proteins and their molecular weights (right panel). (F) GRAS1 RNA enrichment measured by RT-qPCR after immunoprecipitation of NKAP in A549 cells, compared with immunoprecipitation with IgG control antibody; N = 3. (G) Immunoblot of NKAP and p65 in A549 cells treated with control GapmeR compared with GRAS1 GapmeR1 or GRAS1 GapmeR2. (H) Quantification of immunoblot (G). NKAP and p65 expression were calculated after normalization to the GAPDH loading control for each sample; N = 3. (I) RT-qPCR analysis of GCLC, ANGPT1, BDKRB1, and NR3C1 expression levels in A549 cells treated with control GapmeR compared with GRAS1 GapmeR1 or GRAS1 GapmeR2, normalized to GAPDH control mRNA; N = 3. (J) RT-qPCR analysis of NKAP mRNA expression in A549 cells treated with control GapmeR compared with GRAS1 GapmeR1 or GRAS1 GapmeR2, normalized to GAPDH control mRNA; N = 3. (K) Immunoblot of NKAP and p65 in A549 cells treated with control GapmeR compared with GRAS1 GapmeR1 or GRAS1 GapmeR2, with 5 μM MG-132 treatment. (L) Quantification of immunoblot (K). NKAP and p65 expression were calculated after normalization to GAPDH loading control for each sample; N = 6. Error bars represent SD in all panels; (ns) P > 0.05; (***) P < 0.001 by two-tailed t test.

Article Snippet: The following primary antibodies were used: NKAP polyclonal antibody (ab229096; Abcam, 1:1,000 dilution), phospho-histone (γ) H2A.X Ser139 20E3 (60566SF; Cell Signaling Technology, 1:1,000 dilution), p53 (10442-1-AP; Proteintech, 1:1,000 dilution), GAPDH (60004-1-Ig; Proteintech, 1:3,000 dilution), Lamin A/C (10298-1-AP; Proteintech, 1:1,000 dilution), NF-κB p65 (10745-1-AP; Proteintech, 1:1,000 dilution), and TIGA1 (MBS9610109; MyBioSource, 1:1,000 dilution).

Techniques: Purification, Mass Spectrometry, Quantitative RT-PCR, Analysis, Western Blot, Marker, Biomarker Discovery, Control, Positive Control, Luciferase, Negative Control, Immunoprecipitation, Expressing, Two Tailed Test

Journal: CNS Neuroscience & Therapeutics

Article Title: Targeting Microglial HDAC5 Suppresses NLRP3 Inflammation via NF ‐ κB Deacetylation to Alleviate Ischemic Brain Injury

doi: 10.1002/cns.71161

Figure Lengend Snippet: HDAC5 deacetylates NFκB p65 and indirectly regulates NLRP3 expression. (A) BV2 cells were transfected with lentivirus for 48 h and then harvested for Co‐IP assay. (B) Molecular docking analysis predicting the interaction between HDAC5 and NF‐κB p65. The enlarged inset shows the predicted potential binding site. (C) Co‐IP assay showing the interaction between HDAC5 and NF‐κB p65 in BV2 cells. (D) Western blot assay to detect the expression of acetylated proteins after Hdac5 KD. (E) Hdac5 KD in BV2 cells and subsequent Co‐IP assay. (F) Western blot assay to detect NFκB p65 protein expression in cytoplasm and nucleus after Hdac5 KD. (G, H) Quantitative analysis of NFκB p65 protein form (D) ( n = 4). (I) Schematic of the predicted NFκB p65 (Rela) binding site in the promoter of NLRP3. All quantitative data are expressed as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 by one‐way ANOVA.

Article Snippet: Equal amounts of protein were incubated overnight at 4°C with anti‐HDAC5 antibody (Santa Cruz), anti‐acetylated‐lysine antibody (Cell Signaling Technology), anti‐NF‐κB p65 antibody (Santa Cruz), or the corresponding species‐matched IgG as a negative control.

Techniques: Expressing, Transfection, Co-Immunoprecipitation Assay, Docking Assay, Analysis, Binding Assay, Western Blot