Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Western Blot

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: Journal of Alzheimer's Disease Reports

Article Title: Potential link of high fat diet and mRNA expression of Alzheimer's disease-related genes in the enteric mucosa of a rat model of Alzheimer's disease

doi: 10.1177/25424823251358414

Figure Lengend Snippet: Schematic diagram of the study design. Twelve-month-old male and female Wild-type [Fischer 344 (F344); WT] or the amyloid precursor protein/presenilin1 (APP/PS1)-overexpressing Transgenic (TG) rats were used in this study (n = 4 per experimental group). This schematic figure shows the experimental plan for the 12 months old WT and TG rats that were fed Control diet (CD) or high-fat diet (HFD) for following six months. At the end of six months rats were sacrificed, to assess the changes in the mRNA expression of Alzheimer's disease related genes in the ileal mucosa.

Article Snippet: Sections were then incubated in AβPP rabbit monoclonal antibody (APP, E5X 2B, Cat# 2979, Cell Signaling Technology) at 1:200 dilution at room temperature for overnight.

Techniques: Transgenic Assay, Control, Expressing

Journal: Journal of Alzheimer's Disease Reports

Article Title: Potential link of high fat diet and mRNA expression of Alzheimer's disease-related genes in the enteric mucosa of a rat model of Alzheimer's disease

doi: 10.1177/25424823251358414

Figure Lengend Snippet: Demonstration of amyloid-β protein precursor immunohistochemistry in ileal mucosa of intestine tissue of male rats. Sparse amyloid-β protein precursor (AβPP) immunoreactivity [APP (E5X2B) Rabbit mAb that recognizes endogenous levels of human AβPP] was present in the intestine of male non-transgenic animals [M-NTG (or WT); control diet or high fat diet (HFD)] (A-D). However, in male transgenic animals [M-TG; control diet or high fat diet (HFD)], (E-H) widespread immuno-positive staining for AβPP is demonstrated in Auerbach's nerve plexus, located between longitudinal and circular smooth muscle layers of the intestinal wall, as well as multifocal staining in Meissner's nerve plexus, located in the submucosa. Bar magnification (A, C, E, G = 100 µm; B, D, F, H = 50 µm). Arrow indicates AβPP immunostaining in the nerve plexus. Each figure is representative of images from n = 4 animals.

Article Snippet: Sections were then incubated in AβPP rabbit monoclonal antibody (APP, E5X 2B, Cat# 2979, Cell Signaling Technology) at 1:200 dilution at room temperature for overnight.

Techniques: Immunohistochemistry, Transgenic Assay, Control, Staining, Immunostaining

Journal: Journal of Alzheimer's Disease Reports

Article Title: Potential link of high fat diet and mRNA expression of Alzheimer's disease-related genes in the enteric mucosa of a rat model of Alzheimer's disease

doi: 10.1177/25424823251358414

Figure Lengend Snippet: Demonstration of amyloid-β protein precursor immunohistochemistry in ileal mucosa of intestine tissue of female rats. Very scant amyloid-β protein precursor (AβPP) immunoreactivity [APP (E5X2B) Rabbit mAb that recognizes endogenous levels of human AβPP] was present in the intestine of female non-transgenic animals [F-NTG (or WT); control diet or high fat diet (HFD)] (A-D). However, in female transgenic animals [F-TG; control diet or high fat diet (HFD)], (E-H), widespread immuno-positive staining for AβPP is demonstrated in Auerbach's nerve plexus, located between longitudinal and circular smooth muscle layers of the intestinal wall, as well as multifocal staining in Meissner's nerve plexus, located in the submucosa. Bar magnification (A, C, E, G = 100 µm; B, D, F, H = 50 µm). Arrow indicates AβPP immunostaining in the nerve plexus. Each figure is representative of image n = 4 in each experimental group.

Article Snippet: Sections were then incubated in AβPP rabbit monoclonal antibody (APP, E5X 2B, Cat# 2979, Cell Signaling Technology) at 1:200 dilution at room temperature for overnight.

Techniques: Immunohistochemistry, Transgenic Assay, Control, Staining, Immunostaining