d gal foxo4 dri group  (MedChemExpress)

Journal: Food Chemistry: Molecular Sciences

Article Title: Usage of nanobody-beta-galactosidase fusion in immunoassays and its application in detecting a peanut allergen

doi: 10.1016/j.fochms.2026.100357

Figure Lengend Snippet: Tagging a nanobody to the N-terminus of β-gal does not affect its activity. (A) Colonies of dH5α bacteria transformed with a plasmid containing the coding sequence for β-gal with Nb16 fused to its N-terminus via a 4AA (GSHV) linker and plated on an LB/Kan plate with IPTG and X-gal. The plate was photographed 24 h after incubation at 37 °C for 16 h. (B), (C), and (D) are the same as (A), except that the bacteria were induced to express β-gal with Nb16 fused to its N-terminus via a longer flexible peptide (GSGASGSHV), a Strep-tag-containing peptide (GSWSHPQFEKHV), or β-gal with purification tags, respectively.

Article Snippet: Milli-Q water was purified in-house using a Milli-Q Advantage A10 system (Millipore, Bedford, MA, USA) and used throughout. o -Nitrophenyl-β-galactoside (ONPG), Isopropyl β-D-1-thiogalactopyranoside (IPTG), Kanamycin (Kan), and X-Gal were purchased from GoldBio (St Louis, MO, USA).

Techniques: Activity Assay, Bacteria, Transformation Assay, Plasmid Preparation, Sequencing, Incubation, Strep-tag, Purification

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: FOXO4-DRI can effectively maintain vascular function in naturally aging mice. (A) Western blot experiments were conducted and quantitatively analyzed to determine the changes in protein levels of Ki-67, Lamin B, P21, P16, and γ-H2AX in the aortas of naturally aged mice after FOXO4-DRI or PBS treatment. n ≥ 4 per group. (B) RT-qPCR analysis was performed to examine the mRNA levels of Il-1β, Il-6, Cxcl15 , and Tnf-α in the aortas of naturally aged mice after FOXO4-DRI and PBS treatments. n = 3 per group. (C) Representative SA-β-Gal staining and quantitative analysis were performed to determine the number of positive cells in the aortas of naturally aged mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. (D) Representative HE staining and quantitative analysis were performed to evaluate the thickness of the aortas in naturally aged mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. (E) Color Doppler imaging and analysis were performed to detect and analyze the structural and blood flow conditions of the aortas in naturally aged mice after FOXO4-DRI and PBS treatments. n = 6 per group. (F) Representative DHE staining and quantitative analysis were performed to assess the ROS levels in the aortas of naturally aged mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Article Snippet: In the D-Gal + FOXO4-DRI group, mice also received D-galactose injections and, after 4 weeks, were given intraperitoneal injections of FOXO4-DRI (5 mg/kg, MCE, HY-P4157) every 2 days for 4 weeks, followed by tissue collection.

Techniques: Western Blot, Quantitative RT-PCR, Staining, Imaging, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: FOXO4-DRI can effectively maintain vascular function in progeroid mice. (A) Western blot experiments were conducted and quantitatively analyzed to determine the changes in protein levels of Ki-67, Lamin B, P21, P16, and γ-H2AX in the aortas of progeroid mice after FOXO4-DRI or PBS treatment. n ≥ 4 per group. (B) RT-qPCR analysis was performed to examine the mRNA levels of Il-1β , Il-6 , Cxcl15 , and Tnf-α in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. n = 3 per group. (C) Representative SA-β-Gal staining and quantitative analysis were performed to determine the number of positive cells in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. Scale bar = 100 μm. n = 5 per group. (D) Representative HE staining and quantitative analysis were performed to evaluate the thickness in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. Scale bar = 100 μm. n = 5 per group. (E) Color Doppler imaging and analysis were performed to detect and analyze the structural and blood flow conditions in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. n = 5 per group. (F) Representative DHE staining and quantitative analysis were performed to assess the ROS levels in the aortas of progeroid mice after FOXO4-DRI and PBS treatments. n = 5 per group. Scale bar = 100 μm. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Article Snippet: In the D-Gal + FOXO4-DRI group, mice also received D-galactose injections and, after 4 weeks, were given intraperitoneal injections of FOXO4-DRI (5 mg/kg, MCE, HY-P4157) every 2 days for 4 weeks, followed by tissue collection.

Techniques: Western Blot, Quantitative RT-PCR, Staining, Imaging, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: FOXO4-DRI can alleviate endothelial cell senescence induced by OGD. (A) Western blot analysis was conducted to quantify the changes in protein levels of Ki-67, Lamin B, P21, P16, and γ-H2AX in senescent HUVECs following treatment with FOXO4-DRI or PBS, with n ≥ 4 per group. (B) Representative immunofluorescence analysis (green for Ki-67, blue for DAPI) was performed to assess the number of Ki-67 positive HUVECs in different groups, with n = 5 per group and scale bar = 40 μm. (C) Fluorescent staining was carried out to analyze the content and localization changes of P21 and γ-H2AX in different groups, with n = 5 per group and scale bar = 40 μm. (D) Representative SA-β-GAL staining and quantification were performed to evaluate the number of positive cells in different groups, with n = 5 per group and scale bar = 100 μm. (E) Tube formation assays were conducted to analyze relative tube lengths in different groups, with n = 5 per group and scale bar = 400 μm. (F) Representative DHE staining and quantification were carried out to evaluate the number of DHE-positive cells in different groups, with n = 5 per group and scale bar = 100 μm. (G) Scratch assays were performed to assess endothelial cell migration coverage at 0, 12, 24, and 36 h in different groups, with n = 5 per group and scale bar = 400 μm. Data are represented as mean ± SEM. Statistical analysis assessed by 2-way RM ANOVA. (H) RT-qPCR analysis was performed to detect mRNA levels of IL-6 , IL-8 , IL-1β , and TNF-α in different groups, with n = 3 per group. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

Article Snippet: In the D-Gal + FOXO4-DRI group, mice also received D-galactose injections and, after 4 weeks, were given intraperitoneal injections of FOXO4-DRI (5 mg/kg, MCE, HY-P4157) every 2 days for 4 weeks, followed by tissue collection.

Techniques: Western Blot, Immunofluorescence, Staining, Migration, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: FOXO4-DRI can block the interaction between P53 and FOXO4. (A) The FOXO4-P53 interaction was analyzed through co-immunoprecipitation in OGD-treated HUVECs, in different groups, with n = 3 per group. (B) Western blot analysis quantified changes in protein levels of pSer46-P53 and P53 in different groups, with n = 5 per group. (C) Western blot analysis quantified changes in protein levels of pSer46-P53 and P53 in different groups, with n = 5 per group. (D) Western blot analysis was performed to examine the distribution of pSer46-P53 and P53 in the cytoplasm and nucleus of HUVECs in different groups. With n = 5 per group. (E) Subcellular localization of pSer46-P53 in different groups of HUVECs was analyzed using representative immunofluorescence staining (pSer46-P53 in green, DAPI in blue), with n = 5 per group, scale bar = 40 μm. (F) Immunofluorescence detection and analysis of subcellular localization of pSer46-P53 and FOXO4 in aortic sections of naturally aged mice (pSer46-P53 in green, FOXO4 in red, DAPI in blue), with n = 5 per group, scale bar = 20 μm. (G) Immunofluorescence detection and analysis of subcellular localization of pSer46-P53 and FOXO4 in aortic sections of progeroid mice (pSer46-P53 in green, FOXO4 in red, DAPI in blue), with n = 5 per group, scale bar = 20 μm. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: In the D-Gal + FOXO4-DRI group, mice also received D-galactose injections and, after 4 weeks, were given intraperitoneal injections of FOXO4-DRI (5 mg/kg, MCE, HY-P4157) every 2 days for 4 weeks, followed by tissue collection.

Techniques: Blocking Assay, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: FOXO4-DRI promotes apoptosis in senescent cells. (A) Western blot analysis quantified changes in protein levels of BAX, Bcl2, and c-Caspase3 in different groups, with n = 5 per group. (B) Western blot analysis assessed changes in protein levels of BAX, Bcl2, and c-Caspase3 in the aortas of naturally aged mice after FOXO4-DRI or PBS treatment, with n = 5 per group. (C) Representative TUNEL staining and quantitative analysis were conducted to determine the number of positive cells in the aortas of naturally aged mice after FOXO4-DRI and PBS treatments, with n = 5 per group and scale bar = 100 μm. (D) Representative TUNEL staining and quantification evaluated the number of TUNEL-positive cells across different groups with n = 5 per group and scale bar = 20 μm. (E) Flow cytometry analysis was used to quantitatively assess the number of Annexin V-positive cells in different groups, with a sample size of n = 5 per group. Data are presented as mean ± SEM and analyzed using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: In the D-Gal + FOXO4-DRI group, mice also received D-galactose injections and, after 4 weeks, were given intraperitoneal injections of FOXO4-DRI (5 mg/kg, MCE, HY-P4157) every 2 days for 4 weeks, followed by tissue collection.

Techniques: Western Blot, TUNEL Assay, Staining, Flow Cytometry, Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: FOXO4-DRI regulates endothelial cell senescence via the P53 signaling pathway

doi: 10.3389/fbioe.2025.1729166

Figure Lengend Snippet: Schematic illustration of FOXO4-DRI in alleviating endothelial cell senescence aging extends cell survival. This study demonstrates that FOXO4-DRI mitigates senescence by inhibiting the FOXO4-P53 interaction, promoting P53 phosphorylation, and inducing apoptosis in senescent cells, thereby alleviating aging-related symptoms. Content produced with BioRender; license VN28TABHO4.

Article Snippet: In the D-Gal + FOXO4-DRI group, mice also received D-galactose injections and, after 4 weeks, were given intraperitoneal injections of FOXO4-DRI (5 mg/kg, MCE, HY-P4157) every 2 days for 4 weeks, followed by tissue collection.

Techniques: Phospho-proteomics, Produced

Journal: mBio

Article Title: Loss of LafB activity reverses daptomycin resistance in E. faecium

doi: 10.1128/mbio.00715-25

Figure Lengend Snippet: TLC analysis of glycolipids in total membrane lipid extracts from E. faecium . Glycolipid profiles of E. faecium SM1, SM1 lafB T577C, SM1Δ lafB , and SM1 Δ lafB /pAT28:: lafB mutants were compared to WT E. faecium SM1. Purified lipid standards included Gal₂DAG [1,2-diacyl-3-O-(α-D-galactosyl(1→6)-β-D-galactosyl)-sn-glycerol] and Glc ₁ DAG [1,2-diacyl-3-O-(α-D-glucopyranosyl)-sn-glycerol] (Avanti Polar Lipids). Glycolipid species Glc ₁ DAG, Glc ₂ DAG, and Gal ₂ DAG were identified based on migration relative to standards. Additional bands representing unknown lipid species are also indicated. The absence of glycolipid bands in the Δ lafB mutant and their restoration in the complemented strain demonstrates the role of lafB in glycolipid biosynthesis. Data are representative of three independent experiments.

Article Snippet: Approximately 5 μL of each of the two standards (Gal 2 DAG: 1,2-diacyl-3- O -(α- d -galactosyl1-6)-β- d -galactosyl- sn -glycerol Avanti Polar Lipids 840524P, Glc 1 DAG: 1,2-diacyl-3-O-(α-D-glucopyranosyl)-sn-glycerol Avanti Polar Lipids 840522P) and ~15 μL of each sample was spotted on the baseline of a Silica gel 60 TLC plate.

Techniques: Membrane, Purification, Migration, Mutagenesis

Journal: Infection and Immunity

Article Title: Quorum-sensing regulator LsrR modulates avian pathogenic Escherichia coli pathogenicity through direct regulation of cysN

doi: 10.1128/iai.00421-25

Figure Lengend Snippet: ( a ) The LsrR protein was expressed and purified. M: BlueStar Prestained Protein Marker (180 kDa); Line 1: purified LsrR protein flow-through with a size of 60 kDa; Line 2: LsrR positive control; and Line 3: PBS negative control. ( b ) Graphic representation and confirmation of the mutant and complementary strains by PCR. PCR lanes 1–3 confirmed cysN deletion with the cysN internal primer-F/R, and lanes 4–6 confirmed cysN deletion with the cysN external primer-F/R. The primers used for the PCR identification of the cysN gene deletion are also indicated. The amplification of the corresponding target genes indicates success in constructing mutant and complemented strains. ( c ) Bluish colonies on X-gal plates indicated interaction between LsrR and cysN (pUT18C-lsrR-BHT101-pKT25-cysN). In contrast, pUT18C-lsrR-BHT101-pKT25-ompA was used as a negative control (no interaction/whitish colonies) and pUT18C-lsrR-BHT101-pKT25-zip as a positive control (interaction/ bluish colonies). ( d ) LsrR binds to the cysN promoters. The binding ability of LsrR to the cysN promoters was determined by electrophoretic mobility shift assay. An increasing amount of LsrR was incubated with excess Cy5.5-labeled probes. Lane 1: free probe. In each panel, from lanes 2 to 7, the concentration of LsrR was 6, 5, 4, 3, 2, and 2 μM, respectively; the amount of Cy5.5-labeled probes was all 50 fmol (the concentrations were all 5 nM). In lanes 8–9, 200 fmol of unlabeled probes were incubated with the LsrR protein beside the labeled probes. In lane 9, protein FabH was run as a negative control. ( e ) LsrR activates cysN transcription. Relative expression of the cysN gene in the APEC94 strain compared to the APEC94∆lsrR mutant, as determined by qRT-PCR. Gene expression was normalized to the housekeeping gene dnaE , and the relative expression level in the APEC94 was set to 1. Data are presented as the mean ± SEM from three independent biological replicates. Statistical significance was determined by an unpaired t -test (*** P < 0.001). ( f ) DNase I footprinting identified an LsrR-binding site within the cysN promoter. Sequence alignment of this protected region revealed the LsrR conserved binding sequence.

Article Snippet: Following overnight culturing, centrifugation, reconstitution, and PCR detection to confirm the sequence, X-GAL Luria-Bertani (LB) agar (Oxoid, Basingstoke, UK) plates were prepared by adding X-GAL (20 mg/mL).

Techniques: Purification, Marker, Positive Control, Negative Control, Mutagenesis, Amplification, Binding Assay, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Concentration Assay, Expressing, Quantitative RT-PCR, Gene Expression, Footprinting, Sequencing

Journal: Cell reports

Article Title: Potentiation of noncanonical NF-κB signaling and marginal zone B cell expansion by CARD11-mediated regulation of p100 processing

doi: 10.1016/j.celrep.2025.116616

Figure Lengend Snippet: (A) HEK293T cells were co-transfected with indicated nanogram amounts of expression vectors for the indicated proteins. Lysates were immunoblotted using the indicated primary antibodies. The level of p52 normalized to the amount of total p100 protein was measured to calculate processing of p100 relative to the p100 + NIK sample set to 1.0. Levels of phospho-p100 were measured by normalizing to the levels of total p100 to assess phosphorylation of p100 relative to the p100 + NIK sample set to 1.0. Values are depicted below representative blots. See for quantifications. (B) The indicated amounts of expression vectors for the indicated proteins were transiently transfected into IKKα-deficient (IKKα KO) or nontarget control (NT) HEK293T cells. Lysates were immunoblotted using the indicated primary antibodies. (C) Top: the indicated amounts of expression vectors for the indicated proteins were transiently transfected into IKKγ-deficient (IKKγ KO) or nontarget control (NT) HEK293T cells. Lysates were immunoblotted using the indicated primary antibodies. Relative processing of p100 to p52 was calculated as described in (A). Values are depicted below representative blots. See for quantifications. Knockout was confirmed using the indicated antibodies. Bottom: NF-κB luciferase reporter activity measured in IKKγ-deficient (IKKγ KO) or nontarget control (NT) HEK293T cells transiently transfected with Igκ 2 -IFN-LUC, pCSK-LacZ, and the indicated amounts of expression vectors for myc-tagged wild-type CARD11 or C49Y CARD11. The relative NF-κB activity is calculated after β-Gal normalization and as a fold increase over the activity observed with Igκ 2 -IFN-LUC and pCSK-LacZ in the absence of CARD11 expression vector. (D) HEK293T cells were co-transfected with indicated nanogram amounts of expression vectors for FLAG-tagged p100, myc-tagged NIK, and myc-tagged CARD11 variants expressed at roughly equivalent protein levels. Relative processing of p100 to p52 was calculated as described in (A). Values are depicted below representative blots. See for quantifications. (E) HEK293T cells were co-transfected with the indicated nanogram amounts of expression vectors for FLAG-tagged p100, myc-tagged NIK, and myc-tagged CARD11 variants including CARD11ΔID and other domain deletions in the CARD11ΔID background so as to achieve roughly equivalent protein expression. Relative processing of p100 to p52 was calculated as described in (A). NF-κB luciferase reporter activity was measured in HEK293T cells transiently transfected with domain deletion myc-CARD11 variants expressed at equivalent protein levels. The relative NF-κB activity is calculated as described in (C). Quantified data are presented as mean with SD, with individual data points shown from three replicates. All data are representative of three independent experiments. For (C) and (E), ordinary one-way ANOVA with Tukey’s or Dunnett’s multiple comparisons test with single pooled variance was used. Significance definitions are as follows: ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: 4μL of lysate was used to measure luciferase activity using a luciferase assay kit (Promega #E1501) recorded by the luminometer (Berthold Technologies TriStarLB 941) integrating for 10s after a 2s delay, according to the manufacturer’s instructions. β-Galactosidase (β-Gal) activity was determined using 2μL of lysate and a chemiluminescent β-Gal reporter gene assay kit (Takara Bio #631712) according to the manufacturer’s instructions.

Techniques: Transfection, Expressing, Phospho-proteomics, Control, Knock-Out, Luciferase, Activity Assay, Plasmid Preparation