Journal: Alzheimer's Research & Therapy

Article Title: ISX9 activates the Wnt/β-catenin signaling pathway and exerts neuroprotective effects in Alzheimer’s disease

doi: 10.1186/s13195-026-01961-5

Figure Lengend Snippet: ISX9 activates the Wnt/β-catenin signaling pathway in mouse hippocampal neuronal HT22 cells. ( A ) The SuperTOPFlash reporter gene was transfected into HT22 cells, and the cells were treated with solvent control (DMSO) and gradient concentrations of ISX9 (2.5-40 μM) for 24 h. Subsequently, the cells were lysed, and the fluorescence intensity was detected and normalized based on the value of the β-gal. ( B ) HT22 cells were treated with control and gradient concentrations of ISX9 (2.5-40 μM) for 24 h. The expression levels of endogenous β-catenin and activated β-catenin proteins were detected by Western blot. Quantitative analysis was performed by ImageJ and normalized relative to GAPDH values, and results were presented on the right. ( C ) HT22 cells were treated with control and 20 μM ISX9 for 24 h, respectively, followed by precipitation of cell lysates with an anti-AXIN1 antibody. The interaction of AXIN1 with LRP6 or β-catenin was detected by immunoblotting. ( D ) HT22 cells were treated with control or gradient concentrations of ISX9 (2.5-20 μM) for 24 h, respectively. After cell lysis, total RNA was extracted, and cDNA was obtained by reverse transcription. RT-qPCR was performed to detect the mRNA expression levels of Wnt target genes NOTUM, AXIN2 and SURVIVIN. The data shown are the mean ± SD (n = 3). Statistical significance was tested by one-way ANOVA followed by the Tukey test. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicated that there was a significant difference compared with the control group

Article Snippet: Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting with the following antibodies: anti-β-catenin (Santa Cruz Biotechnology, Cat#sc-7963), anti-active β-catenin (Cell Signaling Technology, Cat#8814), anti-GAPDH (Transgen Biotech, Cat#HC301), anti-APP (Proteintech, Cat#27320-1-AP), anti-BACE1 (Proteintech, Cat#12807-1-AP), anti-Aβ (Proteintech, Cat#25524-1-AP), anti-Tau (Proteintech, Cat#10274-1-AP), anti-Phospho-Tau (S404) (Proteintech, Cat#81383-1-RR), anti-ZO-1 (Cell Signaling Technology, Cat#5406), anti-OCCLUDIN (Proteintech, Cat#5506), anti-GLUT1 (Proteintech, Cat#21829-1-AP), anti-LGR5 (Abcam, Cat#ab75732), and anti-SOX2 (Cell Signaling Technology, Cat#23064).

Techniques: Transfection, Solvent, Control, Fluorescence, Expressing, Western Blot, Lysis, Reverse Transcription, Quantitative RT-PCR

Journal: Alzheimer's Research & Therapy

Article Title: ISX9 activates the Wnt/β-catenin signaling pathway and exerts neuroprotective effects in Alzheimer’s disease

doi: 10.1186/s13195-026-01961-5

Figure Lengend Snippet: ISX9 enhances the proliferation, and promotes the expression of stemness-related Wnt target genes in hippocampal neuronal cells. HT22 cells were treated with control or gradient concentrations of ISX9 for 24 h, respectively. The cell viability ( A ) and proliferation ( B ) of HT22 cells were detected by MTT and BrdU assays. ( C ) HT22 cells were treated with control or ISX9 (2.5-20 μM) for 24 h, respectively. The cells were lysed to extract total RNA, which was then subjected to reverse transcription for cDNA synthesis. RT-qPCR was performed to detect the mRNA expression levels of stemness-related Wnt target genes LGR5 and SOX2. ( D - F ) HT22 cells were treated with control and 20 μM of ISX9 for 24 h. Then the cells were fixed and followed by IF staining with DAPI and anti-β-catenin antibody ( D ) or anti-LGR5 antibody ( E ) or anti-SOX2 antibody ( F ). Alexa Fluor 594 conjugated goat anti-mouse IgG antibodies were used as secondary antibodies. Scale bar, 20 μm. The results of the quantitative fluorescence analysis were presented on the right. The data shown are the mean ± SD (n = 3). Statistical significance was tested by one-way ANOVA followed by the Tukey test. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicated that there was a significant difference compared with the control group

Article Snippet: Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting with the following antibodies: anti-β-catenin (Santa Cruz Biotechnology, Cat#sc-7963), anti-active β-catenin (Cell Signaling Technology, Cat#8814), anti-GAPDH (Transgen Biotech, Cat#HC301), anti-APP (Proteintech, Cat#27320-1-AP), anti-BACE1 (Proteintech, Cat#12807-1-AP), anti-Aβ (Proteintech, Cat#25524-1-AP), anti-Tau (Proteintech, Cat#10274-1-AP), anti-Phospho-Tau (S404) (Proteintech, Cat#81383-1-RR), anti-ZO-1 (Cell Signaling Technology, Cat#5406), anti-OCCLUDIN (Proteintech, Cat#5506), anti-GLUT1 (Proteintech, Cat#21829-1-AP), anti-LGR5 (Abcam, Cat#ab75732), and anti-SOX2 (Cell Signaling Technology, Cat#23064).

Techniques: Expressing, Control, Reverse Transcription, cDNA Synthesis, Quantitative RT-PCR, Staining, Fluorescence

Journal: Alzheimer's Research & Therapy

Article Title: ISX9 activates the Wnt/β-catenin signaling pathway and exerts neuroprotective effects in Alzheimer’s disease

doi: 10.1186/s13195-026-01961-5

Figure Lengend Snippet: ISX9 activates Wnt/β-catenin signaling in the hippocampus of AD mice. ( A ) Expression levels of total β-catenin and activated β-catenin protein in hippocampal tissues of WT, WT/ISX9, AD and AD/ISX9 group mice. The below bars show the results of quantification of the immunoblotting images by ImageJ and normalization relative to GAPDH values. ( B ) Protein was extracted from the hippocampal tissues of mice, followed by precipitation with an anti-AXIN1 antibody. The interaction of AXIN1 with LRP6 or β-catenin was detected by immunoblotting. ( C ) Total RNA extracted from mouse hippocampal tissues of mice was reverse transcribed to obtain cDNA, and then RT-qPCR was performed to detect the mRNA expression levels of Wnt target genes NOTUM, AXIN2 and SURVIVIN

Article Snippet: Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting with the following antibodies: anti-β-catenin (Santa Cruz Biotechnology, Cat#sc-7963), anti-active β-catenin (Cell Signaling Technology, Cat#8814), anti-GAPDH (Transgen Biotech, Cat#HC301), anti-APP (Proteintech, Cat#27320-1-AP), anti-BACE1 (Proteintech, Cat#12807-1-AP), anti-Aβ (Proteintech, Cat#25524-1-AP), anti-Tau (Proteintech, Cat#10274-1-AP), anti-Phospho-Tau (S404) (Proteintech, Cat#81383-1-RR), anti-ZO-1 (Cell Signaling Technology, Cat#5406), anti-OCCLUDIN (Proteintech, Cat#5506), anti-GLUT1 (Proteintech, Cat#21829-1-AP), anti-LGR5 (Abcam, Cat#ab75732), and anti-SOX2 (Cell Signaling Technology, Cat#23064).

Techniques: Expressing, Western Blot, Reverse Transcription, Quantitative RT-PCR

Journal: Cell Death Discovery

Article Title: Pregnane X receptor protects against age-related bone loss in males via PI3K/Akt-mediated inhibition of apoptosis

doi: 10.1038/s41420-025-02797-y

Figure Lengend Snippet: A Schematic diagram of experimental design. B ELISA for inflammation factors Tnfα, Il1β, and Il6 in the conditional medium collected from Nr1i2 knockdown primary BMSCs or siNC as control. C ELISA for ROS clearance-related enzyme T-SOD, GSH-PX and ROS damage biomarker MDA in Nr1i2 knockdown primary BMSCs or siNC as control. D Flow cytometry analysis of intracellular ROS level by using the fluorescent dye DCFDA. Western blot for PI3K/Akt pathway- ( E ) and apoptosis- ( F ) related proteins in primary BMSCs of different groups. Data were means ± s.e.m. n = 3 independent repeats for in vitro experiments. ** p < 0.01, *** p < 0.001 by t-test.

Article Snippet: The Il1β, Il6, and Tnfα level in the conditional medium, intracellular level of T-SOD, GSH-PX, MDA, and ROS were determined by using the Mouse IL-1β ELISA Kit (HJ177, Epizyme Biotech, Shanghai, China), Mouse IL-6 ELISA Kit (HJ182, Epizyme Biotech, Shanghai, China), and Mouse TNF-α ELISA Kit (HJ207, Epizyme Biotech, Shanghai, China), Total superoxide dismutase (SOD) assay kit (A001-3-2, Jiancheng, Nanjing, China), Glutathione Peroxidase (GSH-PX) Assay Kit (A005-1-2, Jiancheng, Nanjing, China), Malondialdehyde (MDA) assay kit (A003-1-2, Jiancheng, Nanjing, China), and DCFDA/H2DCFDA-Cellular ROS Assay Kit (ab113851, abcam, Cambridge, UK) according to the manufacturer’s instructions, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Knockdown, Control, Biomarker Discovery, Flow Cytometry, Western Blot, In Vitro

Journal: Scientific Reports

Article Title: Recombinant expression, characterization, and quantification in human cancer cell lines of the Anaplastic Large-Cell Lymphoma-characteristic NPM-ALK fusion protein

doi: 10.1038/s41598-020-61936-w

Figure Lengend Snippet: Effect of different lysis reagents on NPM-ALK protein ELISA detection in whole cell extracts. NPM-ALK-expressing Karpas 299 lymphoma cells were lysed with various lysis reagents. Serial dilutions of the cell extracts were tested for the released NPM-ALK protein using the PathScan Total ALK ELISA kit (CST #7322). Total protein content in the whole cell lysates was measured by the bicinchoninic acid (BCA) method against a BSA standard curve and was estimated to be in the range of 100–160 pg of protein per cell.

Article Snippet: We confirmed the reproducibility of sample preparation and the uniformity of cell numbers used per assay using the PathScan Total β-Actin Sandwich ELISA Antibody Pair #7881 (Cell Signaling Technology; capture: β-actin rabbit monoclonal antibody; detection: pan-actin mouse monoclonal antibody; HRP-linked anti-mouse secondary IgG antibody). β-actin was used to confirm the ELISA results by 2-D electrophoresis and also can be potentially used for later normalization of number of cells in biopsy specimens.

Techniques: Lysis, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Scientific Reports

Article Title: Recombinant expression, characterization, and quantification in human cancer cell lines of the Anaplastic Large-Cell Lymphoma-characteristic NPM-ALK fusion protein

doi: 10.1038/s41598-020-61936-w

Figure Lengend Snippet: ( A ) ELISA characterization with recombinant NPM-ALK fusion protein. Two-fold serial dilutions of NPM-ALK recombinant protein were spiked in PBS buffer or Cell Lysis Reagent and used in the ELISA with the ab180607 (capture) /#3791 (detection) antibody pair (n = 3, average ± 1 SD). An HRP-conjugated horse anti-mouse IgG antibody (#7076) from Cell Signaling Technology along with 1-Step™ Ultra TMB-ELISA Substrate Solution was used for signal development. (Inset) The fitted 5-point linear calibration curve is shown with the red dotted line; estimated LOD is shown with the black dotted line. ( B ) NPM-ALK ELISA specificity . Immunodetection of cellular NPM-ALK protein in cultured human lymphoma (Karpas 299, SU-DHL-1, Jurkat, U937) and neuroblastoma (IMR-32) cells by ELISA with the ab180607 (capture) /#3791 (detection) antibody pair (n = 3, average ± 1 SD). Whole cell extracts were prepared in Cell Lysis Buffer. An HRP-conjugated horse anti-mouse IgG antibody (#7076) from Cell Signaling Technology along with 1-Step™ Ultra TMB-ELISA Substrate Solution was used for signal development.

Article Snippet: We confirmed the reproducibility of sample preparation and the uniformity of cell numbers used per assay using the PathScan Total β-Actin Sandwich ELISA Antibody Pair #7881 (Cell Signaling Technology; capture: β-actin rabbit monoclonal antibody; detection: pan-actin mouse monoclonal antibody; HRP-linked anti-mouse secondary IgG antibody). β-actin was used to confirm the ELISA results by 2-D electrophoresis and also can be potentially used for later normalization of number of cells in biopsy specimens.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Lysis, Immunodetection, Cell Culture