Journal: Acta Pharmaceutica Sinica. B

Article Title: Steroid hormone ouabain cooperates with Na + ,K + -ATPase to improve innate immunity against bacterial infections through a non-classic training mechanism regulated by mRNA reprogramming

doi: 10.1016/j.apsb.2025.12.041

Figure Lengend Snippet: PKC α -dependent HuR nuclear export is involved in the ouabain’s training effect. (A) The tolerant THP1 cells were treated with 50 nmol/L ouabain at different times. The expression of cytoplasmic or nuclear HuR was examined by Western blot analysis, and the quantification results are shown on the right, n = 3. (B) THP1 cells were trained with 50 nmol/L ouabain for 24 h, then recovered for 1–5 days. The expression of cytoplasmic and nuclear HuR was examined. (C) The cytoplasmic and nuclear HuR expressions were examined in PMs from NKA α1 +/+ and NKA α1 +/− mice ( n = 3). (D) PMs from HuR WT and HuR −/− RAW264.7 cells were trained with ouabain, n = 6. (E) Groups of mice were treated with liposomes to deplete macrophages, followed by a moderate CLP and S.tm. (2 × 10 3 CFU, i.p.) infection (72 h post-CLP). At 48 h post-CLP, mice were adoptively transferred with ouabain-trained HuR WT or HuR −/− RAW264.7 cells, and the treatments were repeated three times at 72, 96, and 120 h post-CLP, with n = 10 mice per group. (F) THP1 cells were pre-treated with the SB203580 (p38 MAPK inhibitor), Gö6983 (protein kinase C inhibitor), BAY11-7082 (IkB kinase α inhibitor), and GSK-3 β inhibitor 1 for 2 h. The expression of cytoplasmic or nuclear HuR was examined. (G) THP1 cells were transfected with si-NC or si-PKC α for 36 h and then incubated with or without 50 nmol/L ouabain for 12 h. The expression of cytoplasmic and nuclear PKC α or HuR was examined. (H) The HEK293T cells, after transfection with a TNF 3′-UTR luciferase reporter and siRNAs against PKC α , were treated with 50 nmol/L ouabain for 12 h, and the luciferase activity of the si-NC group was set at 1.0 ( n = 3). (I) THP1 cells were treated with ouabain at 50 nmol/L for different periods, and the protein expression of p-PKC α , PKC α , and HuR in cytoplasmic and nuclear fractions were examined. (J) THP1 cells were treated with 50 nmol/L ouabain for 12 h, and the protein lysates of nuclear fractions were immunoprecipitated with control IgG or anti-HuR IgG overnight. The protein expressions of PKC α and HuR in the immunoprecipitate were examined. (K) THP1 cells were treated with 50 nmol/L ouabain for the indicated periods. Cells were incubated with Fluo-4 AM for 60 min at 37 °C, and the fluorescence intensity in the FL1 channel was detected using a flow cytometer. (L) THP1 cells were pre-treated with the calcium chelator BAPTA-AM (10 μmol/L) for 2 h before the addition of ouabain (50 nmol/L). (M) The HEK293T cells were transfected with Flag-tagged HuR and its mutants for 24 h, then treated with or without 50 nmol/L ouabain for 12 h. HuR expressions in the cytoplasmic and nucleus fractions were examined. (N) The HEK293T cells were transfected with the indicated constructs, and luciferase activity was measured using a dual-luciferase reporter assay ( n = 3). (O) The tolerant THP1 cells were treated with 50 nmol/L ouabain for different periods, and the protein expressions of p-PKC α and PKC α were examined. (P) The tolerant THP1 cells were treated with ouabain at the indicated concentrations for 4 h, n = 3.

Article Snippet: Ouabain octahydrate (purity 99%, HY-B0542), β - d -glucan (HY-139413), anti-mouse PD-L1 Ab (HY- P99145 ), protein A/G magnetic beads (HY-K0202), streptavidin magnetic beads (HY-K0208), Gö 6983 (HY-13689), GSK-3 β inhibitor 1 (HY-126144), SB203580 (HY-10256), BAY11-7082 (HY-13453), and erlotinib (HY-50896) were all purchased from MedChemExpress (NJ, USA).

Techniques: Expressing, Western Blot, Liposomes, Infection, Transfection, Incubation, Luciferase, Activity Assay, Immunoprecipitation, Control, Fluorescence, Flow Cytometry, Construct, Reporter Assay

Journal: Molecular and Cellular Biology

Article Title: Muscle-Specific Deletion of the Glut4 Glucose Transporter Alters Multiple Regulatory Steps in Glycogen Metabolism

doi: 10.1128/mcb.25.21.9713-9723.2005

Figure Lengend Snippet: FIG. 4. Basal IRS-1-associated PI3K (A), Akt (B), and GSK3 (C) activity levels in muscle from WT and muscle-G4KO mice. Mice were fasted overnight. (A) PI3K activities were measured in IRS-1 immunoprecipitates and were quantitated using a PhosphorImager. (B) Muscle lysates (500 g) were subjected to immunoprecipitation with an Akt antibody that recognizes both Akt1 and Akt2. The immune pellets were assayed for kinase activity using Crosstide as the substrate. (C) Muscle lysates (500 g) were subjected to immunoprecipitation with a GSK3-specific antibody. The immune pellets were assayed for kinase activity using phospho-glycogen synthase 1 as the substrate. Results are means SEM for four to six mice per group. *, P 0.05 versus WT.

Article Snippet: The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with the following antibodies in 1% nonfat dry milk overnight at 4°C: a polyclonal Glut4 antibody (gift from H. Haspel, Henry Ford Hospital, Detroit, MI), a polyclonal insulin receptor antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a polyclonal IRS-1 antibody (gift from M. White, Joslin Diabetes Center), a polyclonal antibody against the p85 subunit or p110 subunit of PI3K (Upstate Biotechnology), a polyclonal antibody for Akt that recognizes both Akt1 and Akt2 (Upstate Biotechnology), a monoclonal antibody for GSK3 that recognizes both GSK3 and GSK3 (Upstate Biotechnology), a polyclonal glycogen synthase antibody (gift from J. Lawerence, University of Virginia), a polyclonal mitogen-activated protein kinase (MAPK) antibody (gift from J. Blenis, Harvard Medical School), a polyclonal PP1 antibody (Upstate Biotechnology), a polyclonal RGL/GM antibody (gift from A. DePaoli-Roach, Indiana University) a polyclonal PTG antibody (gift from M. Brady, University of Chicago), a polyclonal phosphoglycogen synthase (Ser641) antibody (Cell Signaling, Beverly, MA), or a polyclonal hexokinase II (Chemicon, Temecula, CA).

Techniques: Activity Assay, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Muscle-Specific Deletion of the Glut4 Glucose Transporter Alters Multiple Regulatory Steps in Glycogen Metabolism

doi: 10.1128/mcb.25.21.9713-9723.2005

Figure Lengend Snippet: FIG. 5. Insulin-stimulated IRS-1-associated PI3K (A), Akt (B), GSK3 (C), and glycogen synthase (D) activity levels in muscle from WT and muscle-G4KO mice. After an overnight fast, mice were injected i.v. with saline (, white bars) or 10 U/kg insulin (, black bars). Three min later, muscle was removed. (A) PI3K activities were measured in IRS-1 immunoprecipitates and were quantitated using a PhosphorImager. (B) Muscle lysates (500 g) were subjected to immunoprecipitation with an Akt antibody that recognizes both Akt1 and Akt2. The immune pellets were assayed for kinase activity using Crosstide as the substrate. (C) Muscle lysates (500 g) were subjected to immunoprecipitation with a GSK3- specific antibody. The immune pellets were assayed for kinase activity using phospho-glycan synthase 1 as the substrate. (D) The ratio of glycogen synthase (GS) activity represents the activity measured in the absence divided by that in the presence of glucose-6-phosphate. Results are means SEM for four to six mice per group. *, P 0.05 versus insulin-stimulated WT.

Article Snippet: The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with the following antibodies in 1% nonfat dry milk overnight at 4°C: a polyclonal Glut4 antibody (gift from H. Haspel, Henry Ford Hospital, Detroit, MI), a polyclonal insulin receptor antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a polyclonal IRS-1 antibody (gift from M. White, Joslin Diabetes Center), a polyclonal antibody against the p85 subunit or p110 subunit of PI3K (Upstate Biotechnology), a polyclonal antibody for Akt that recognizes both Akt1 and Akt2 (Upstate Biotechnology), a monoclonal antibody for GSK3 that recognizes both GSK3 and GSK3 (Upstate Biotechnology), a polyclonal glycogen synthase antibody (gift from J. Lawerence, University of Virginia), a polyclonal mitogen-activated protein kinase (MAPK) antibody (gift from J. Blenis, Harvard Medical School), a polyclonal PP1 antibody (Upstate Biotechnology), a polyclonal RGL/GM antibody (gift from A. DePaoli-Roach, Indiana University) a polyclonal PTG antibody (gift from M. Brady, University of Chicago), a polyclonal phosphoglycogen synthase (Ser641) antibody (Cell Signaling, Beverly, MA), or a polyclonal hexokinase II (Chemicon, Temecula, CA).

Techniques: Activity Assay, Injection, Saline, Immunoprecipitation, Glycoproteomics

Journal: Molecular and Cellular Biology

Article Title: Muscle-Specific Deletion of the Glut4 Glucose Transporter Alters Multiple Regulatory Steps in Glycogen Metabolism

doi: 10.1128/mcb.25.21.9713-9723.2005

Figure Lengend Snippet: FIG. 9. Signaling protein levels in muscle from WT and muscle- G4KO mice. Mice were fasted overnight. Proteins in muscle lysates (25 to 50 g) were separated by SDS-PAGE on 6, 8, or 10% gels and transferred to nitrocellulose membranes. Insulin receptor (IR), IRS-1, p110, p85, Akt, GSK3/, and MAPK were visualized by immuno- blotting with specific antibodies. This blot is representative of three blots on six to nine mice per group.

Article Snippet: The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with the following antibodies in 1% nonfat dry milk overnight at 4°C: a polyclonal Glut4 antibody (gift from H. Haspel, Henry Ford Hospital, Detroit, MI), a polyclonal insulin receptor antibody (Santa Cruz Biotechnology, Santa Cruz, CA), a polyclonal IRS-1 antibody (gift from M. White, Joslin Diabetes Center), a polyclonal antibody against the p85 subunit or p110 subunit of PI3K (Upstate Biotechnology), a polyclonal antibody for Akt that recognizes both Akt1 and Akt2 (Upstate Biotechnology), a monoclonal antibody for GSK3 that recognizes both GSK3 and GSK3 (Upstate Biotechnology), a polyclonal glycogen synthase antibody (gift from J. Lawerence, University of Virginia), a polyclonal mitogen-activated protein kinase (MAPK) antibody (gift from J. Blenis, Harvard Medical School), a polyclonal PP1 antibody (Upstate Biotechnology), a polyclonal RGL/GM antibody (gift from A. DePaoli-Roach, Indiana University) a polyclonal PTG antibody (gift from M. Brady, University of Chicago), a polyclonal phosphoglycogen synthase (Ser641) antibody (Cell Signaling, Beverly, MA), or a polyclonal hexokinase II (Chemicon, Temecula, CA).

Techniques: SDS Page

Journal: Molecular Cancer

Article Title: DKK1 promotes hepatocellular carcinoma cell migration and invasion through β-catenin/MMP7 signaling pathway

doi: 10.1186/1476-4598-12-157

Figure Lengend Snippet: Beta-catenin is involved in the dual promotive function of DKK1 on tumor migration and invasion. A : Knockdown of DKK1 repressed the mRNA and protein level of β-catenin in Bel7402 cells. B : Silencing of DKK1 attenuated the β-catenin-dependent transcriptional activity. Tumor cells were transfected with firefly luciferase reporter plasmids containing either wild-type or mutant TCF binding sites (indicated as Wt or Mut on the X axis), and a Renilla luciferase expressing construct. The firefly luciferase activity of each sample was normalized to the Renilla luciferase activity. The normalized luciferase activity of shControl-transfectants in each experiment was set as relative luciferase activity 1. Therefore, no error bar is shown for shControl-transfectants. **, p < 0.01, compared with shControl-transfectants. C : Transient expression of β-catenin in Bel7402-shDKK1 cells was detected by RT-PCR analysis. Bel7402-shDKK1 cells transfected with pcDNA3 (lane 2) or pcDNA3-β-catenin (lane 3) were analyzed using RT-PCR. D : Restoration of β-catenin reversed the anti-invasion effect of shDKK1. E, F : Treatment with GSK3β inhibitor LiCl (5 mM) attenuated the anti-migration and anti-invasion effect of shDKK1. In (E) , after scratching a wound and removing the floating cells, tumors cells without treatment (panel 1, 3) or incubated with 5 mM LiCl (panel 2, 4) were applied to the wound scratch assay for 24 h. In (F) tumors cells treated as in (E) were applied to transwell chamber coated with Matrigel and then incubated for 24 h. The data are represented as means ± SEM. *, p <0.05; **, p <0.01.

Article Snippet: The sources of primary antibodies used for Western blot: DKK1 (cat. sc-25516), LRP6 (cat. sc-17982), GSK3β (cat. sc-53931), β-catenin (cat. sc-7199) and MMP7 (cat. sc-101566) as well as the corresponding horseradish peroxidase-conjugated second antibodies were all purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA).

Techniques: Migration, Knockdown, Activity Assay, Transfection, Luciferase, Mutagenesis, Binding Assay, Expressing, Construct, Reverse Transcription Polymerase Chain Reaction, Incubation, Wound Healing Assay

Journal: Molecular Cancer

Article Title: DKK1 promotes hepatocellular carcinoma cell migration and invasion through β-catenin/MMP7 signaling pathway

doi: 10.1186/1476-4598-12-157

Figure Lengend Snippet: DKK1 regulates β-catenin expression through the non-canonical Wnt pathway. A : Restoration of DKK1 promoted the mRNA expression of β-catenin in HepG2 cells. B : Knockdown of DKK1 attenuated β-catenin mRNA expression in Bel7402 cells. C : Restoration of DKK1 promoted the protein levels of β-catenin and c-Myc in HepG2 cells. D : Knockdown of DKK1 attenuated the protein levels of β-catenin and c-Myc in Bel7402 cells. E : IWP-2 attenuated the positive effect of DKK1 on β-catenin expression in HepG2 cells. F : Treatment with GSK3β inhibitor LiCl restored the expression of β-catenin in Bel7402 cells.

Article Snippet: The sources of primary antibodies used for Western blot: DKK1 (cat. sc-25516), LRP6 (cat. sc-17982), GSK3β (cat. sc-53931), β-catenin (cat. sc-7199) and MMP7 (cat. sc-101566) as well as the corresponding horseradish peroxidase-conjugated second antibodies were all purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA).

Techniques: Expressing, Knockdown

Journal: Journal of Clinical Investigation

Article Title: JAM3 maintains leukemia-initiating cell self-renewal through LRP5/AKT/β-catenin/CCND1 signaling

doi: 10.1172/jci93198

Figure Lengend Snippet: Figure 5. LRP5 interacts with PDK1 to activate AKT signaling to inhibit GSK3β. (A) Protein levels of phospho-PDK1 (S241), PDK1, phospho-AKT (T308), AKT, phospho-GSK3β (S9), and GSK3β were measured in WT and Jam3-null YFP+Mac-1+c-Kit+ LICs by immunoblotting. (B) V5-tagged PDK1 and FLAG- tagged LRP5 were overexpressed in 293T cells, and their lysates were coimmunoprecipitated by V5 antibodies and protein A/G beads, followed by Western blotting analysis for FLAG (LRP5). (C) A reverse coimmunoprecipitation experiment was performed after LRP5-FLAG pull-down, followed by Western blotting analysis for PDK1 (V5). (D) A constitutively active form of phospho-AKT (E17K, AKTCN) was subcloned into pCDH-EF1a-T2A-mCherry vector and ectopically expressed in Jam3-null leukemia cells, followed by injection into recipient mice. Survival was compared among the mice receiving WT cells, Jam3-null cells, and AKTCN-overexpressing WT or Jam3-null cells (n = 5–7; **P < 0.01, ***P < 0.001, log-rank test). (E) Phospho-AKT (T308) and AKT levels were validated in leukemia cells from the rescue experiment in D. (F) The cell cycle distribution in YFP+Mac-1+c-Kit+ LICs from the rescue experiment in D was determined using Ki-67 and Hoechst 33342 staining (n = 3; ***P < 0.001, 2-way ANOVA followed by Bonferroni’s post-test). The empty vector was used as the control. Experiments were conducted 3 times for validation.

Article Snippet: The primary antibodies were as follows: anti–human strepII (GenScript), anti–human V5 (Biodragon), anti–human FLAG (Cell Signaling Technology), anti-CCND1 (Cell Signaling Technology), anti-GSK3β (Cell Signaling Technology), anti–phospho-GSK3β (S9) (Abways), anti–β-catenin (Proteintech), anti–phospho–β-catenin (S552) (Cell Signaling Technology), anti-AKT (Santa Cruz Biotechnology), anti–phospho-AKT (T308) (Abways), anti-PDK1 (Bioworld), anti–phospho-PDK1 (S241) (Bioworld), anti-LRP5 (Cell Signaling Technology), and anti–β-actin (Calbiochem).

Techniques: Western Blot, Plasmid Preparation, Injection, Staining, Control, Biomarker Discovery

Journal: Cells

Article Title: Extract of Curculigo capitulata Ameliorates Postmenopausal Osteoporosis by Promoting Osteoblast Proliferation and Differentiation.

doi: 10.3390/cells13232028

Figure Lengend Snippet: Figure 8. The Eocc activates the Src/PI3K/AKT and downstream signaling pathways via ERα: (A,B) Western blot analysis and the quantification of Src, p-Src, PI3K, p-PI3K, AKT, p-AKT, cleaved- caspase-3, and cleaved-caspase-9 expression levels. (C,D) Western blot analysis and quantification of mTOR, p-mTOR, p70S6K, and p-p70S6K expression levels. (E,F) Western blot analysis and quan- tification of GSK3β and p-GSK3β expression levels. (G,H) Representative images and quantitative analysis of cell apoptosis rates measured by flow cytometry. All bar graphs represent mean ± SD. Comparisons versus the control group: * p < 0.05, ** p < 0.01, *** p < 0.001; versus the normal group: ### p < 0.001; & p < 0.05, && p < 0.01, &&& p < 0.001.

Article Snippet: After blocking with 5% BSA at room temperature for 2 h, each blot was incubated with primary antibodies against Osteocalcin (OCN) (Wanlei bio, WLH4378, 1:1000), Runx2 (db16015, 1:1000), Osteopontin (OPN) (db15106, 1:1000) and BSP (db16122, 1:1000) sourced from Diagbio, β-actin (AF7018, 1:1000), ERα (AF6058, 1:1000), p-ERα (AF3061, 1:1000), Akt (AF6261, 1:1000), p-AKT (AF0016, 1:1000), p-PI3K (AF3241, 1:1000), cleaved-caspase-9 (AF5240, 1:1000), and cleaved-caspase-3 (AF7022, 1:1000) purchased from Affinity, PI3K (A23303, 1:1000), GSK3β (A2081, 1:1000), p-GSK3β (AP1088, 1:1000), mTOR (A25581, 1:1000), p-mTOR (AP0115, 1:1000), Src (A0324, 1:1000), p-Src (AP1027, 1:1000), p70S6K (A2190, 1:1000), and p-p70S6K (AP0564, 1:1000) purchased from Abclonal.

Techniques: Protein-Protein interactions, Western Blot, Expressing, Flow Cytometry, Control

Journal: Experimental & Molecular Medicine

Article Title: Canonical Wnt signaling regulates Mbd3 protein stability during neurogenesis

doi: 10.1038/s12276-025-01510-4

Figure Lengend Snippet: a Immunostaining detecting β-catenin and Mbd3 enrichment in undifferentiated NPCs and 2DIV differentiated NPCs treated with GSK3β inhibitor (CHIR). Nuclear staining is shown by DAPI (blue). Scale bars, 100 µm (black) and 50 µm (yellow). b , c Quantification of protein levels of β-catenin ( b ) and Mbd3 ( c ) in undifferentiated and 2DIV differentiated NPCs. d A plot of TOP/FOP Flash assay indicating β-catenin levels through luciferase activity in CHIR-treated differentiated NPCs. e Time-dependent levels of β-catenin and Mbd3 protein in HEK293T cells following CHX and with or without CHIR treatment ( N = 3). Labels a–c denote Mbd3 isoforms. f , g Corresponding quantification of protein levels of β-catenin ( f ) and Mbd3 ( g ). h IP of HA-Ub in the NPCs expressing full-length Myc-Mbd3 with control or V5 tagged GSK3β (V5-GSK3β) expression vector ( N = 3). IP was demonstrated using an anti-Myc antibody. Overexpression of GSK3β was shown through anti-V5 antibody. i Corresponding relative level of Mbd3 ubiquitination in GSK3β overexpression. j Myc-Mbd3 and HA-Ub expression vectors were transfected to NPCs carrying control siRNA or GSK3β siRNA and subjected to co-IP with anti-Myc antibody ( N > 3). k Corresponding relative level of Mbd3 ubiquitination in GSK3β silence. l IP of HA-Ub in the NPCs expressing full-length Myc-tagged Mbd3 (Myc-Mbd3) with or without CHIR treatment ( N = 3). m Corresponding relative level of Mbd3 ubiquitination in GSK3β overexpression induced by CHIR. In all cases, data are presented as mean ± s.d. Statistical significance was determined using an unpaired two-tailed Student’s t -test for b – d (* P < 0.05, ** P < 0.01, *** P < 0.001) and one-way ANOVA for f , g , i , k and m (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: To deplete specific target genes, GSK3β siRNA (sc-35525, Santa Cruz Biotechnology), Mbd3 siRNA (sc-35868, Santa Cruz Biotechnology) and HDAC1 siRNA (sc-29344, Santa Cruz Biotechnology) were used and tested against a control siRNA construct (sc-37007).

Techniques: Immunostaining, Staining, Luciferase, Activity Assay, Expressing, Control, Plasmid Preparation, Over Expression, Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay, Two Tailed Test