Journal: Neurobiology of disease

Article Title: Upregulation of Integrin beta-3 in astrocytes upon Alzheimer's disease progression in the 5xFAD mouse model.

doi: 10.1016/j.nbd.2024.106410

Figure Lengend Snippet: Fig. 2. Relative comparison of ITGB3, GFAP and p-FAK(Tyr397)/FAK proteins and Itgb3 mRNA changes in cortex and hippocampus of WT and 5xFAD mice. Analysis of the WB bands normalized to β-actin in protein samples from WT and 5xFAD cortex and hippocampus at A) 2 months of age, B) 6 months of age and C) 12 months of age. Ratios of p-FAK/FAK were obtained from phosphorylated protein bands after normalization with intensities of total FAK. Representative WB bands of ITGB3, GFAP, total FAK, p-FAK(Tyr397) and β-actin from cortical and hippocampal protein samples of WT and 5xFAD mice from the age of D) 2 months, E) 6 months and F) 12 months. Comparison of normalized to Gapdh fold Itgb3 mRNA expression in cortical and hippocampal samples from WT and 5xFAD cortex and hippocampus at G) 2 months of age, H) 6 months of age I) 12 months of age. All data are presented as relative to age-matched WT-group mean (±SEM) followed by unpaired t-test comparison to measure the genotype effect between WT and 5xFAD mice, n = 4–6 mice per group. «*» - Genotype-related effect: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The membranes were incubated at +4 ◦C overnight with the primary rabbit antibodies against ITGB3 (1:1000; #13166, Cell Signaling Technology, Danvers, MA, USA), FAK (Focal adhesion kinase; 1:1000; #3285, Cell Signaling Technology), Phospho-FAK (Tyr397) (1:1000; #3283, Cell Signaling Technology), GFAP (1:1000; #Z033429–2, DAKO, Agilent Technologies, USA).

Techniques: Comparison, Expressing

Journal: Neurobiology of disease

Article Title: Upregulation of Integrin beta-3 in astrocytes upon Alzheimer's disease progression in the 5xFAD mouse model.

doi: 10.1016/j.nbd.2024.106410

Figure Lengend Snippet: Fig. 4. Aβ-mediated alterations in ITGB3 expression and signaling in adult astrocytes. Murine primary adult astrocytes were treated with 5 μM Aβ1–42 for 24 h prior sampling. Bar Chart (A) represents the signal intensity of astrocytic LDH release and is expressed as a percentage of Vehicle group as control values (100%). (B) Fold- changes of Itgb3 and Gfap mRNA normalized to Gapdh after Aβ1–42 treatment. (C) Analysis and representative images of the ITGB3, GFAP and p-FAK/FAK WB bands normalized to β-actin after incubation with Aβ1–42. All data are presented as relative to control group (Vehicle) mean (±SEM) followed by paired Two-tailed t-test comparison to measure treatment effect: *p < 0.05. Data is representative of 4 independent experiments (n = 4).

Article Snippet: The membranes were incubated at +4 ◦C overnight with the primary rabbit antibodies against ITGB3 (1:1000; #13166, Cell Signaling Technology, Danvers, MA, USA), FAK (Focal adhesion kinase; 1:1000; #3285, Cell Signaling Technology), Phospho-FAK (Tyr397) (1:1000; #3283, Cell Signaling Technology), GFAP (1:1000; #Z033429–2, DAKO, Agilent Technologies, USA).

Techniques: Expressing, Sampling, Control, Incubation, Two Tailed Test, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: MFG-E8 attenuates intestinal inflammation in murine experimental colitis by modulating osteopontin-dependent alphavbeta3 integrin signaling.

doi: 10.4049/jimmunol.0803711

Figure Lengend Snippet: FIGURE 9. Effects of MFG-E8 on focal adhesion ki- nase in LPS-treated peritoneal macrophages. A, Western blotting was performed using cultured peritoneal mac- rophages treated with LPS (100 ng/ml) for various time points with the anti-p3 integrin (pY759) Ab from 3 integrin immunoprecipitated samples and anti-pFAK Ab from FAK immunoprecipitated samples. B, Western blotting results showing pFAK among total FAK in peritoneal macrophages treated with LPS, OPN, or MFG-E8 proteins for the indicated time periods. Blots shown are representative of three independent experi- ments. C, Proposed model of MFG-E8-mediated antiin- flammatory effects in immune-reactive cells. An exter- nal stimulus factor, such as LPS, activates inflammatory cascades via TLR-4-mediated pathways by utilizing a variety of signaling events. Although LPS-mediated NF-B induction is common, it also activates FAK and subsequently v3 integrin to generate inside-out sig- naling for enhanced binding of its potential ligands. OPN, which becomes activated during colitis or in an LPS-treated condition, recognizes activated v3 inte- grin and performs outside-in signaling via FAK phos- phorylation. Exogenous MFG-E8 interferes with OPN binding to v3 integrin and affects downstream signal- ing for NF-B activation by modulating FAK phosphorylation.

Article Snippet: The Abs used were: anti-mouse MFG-E8 (MBL International), PE-F4/80 (eBioscience), anti-His probe (H3), anti-p-I B, anti- 3 integrin, anti-focal adhesion kinase (FAK), and anti-pFAK (Santa Cruz Biotechnology), antiI B, anti-OPN, and anti-IgG (R&D Systems), anti- v integrin (Abcam), and anti-phospho- 3 (pY759) integrin (BD Biosciences).

Techniques: Western Blot, Cell Culture, Immunoprecipitation, Binding Assay, Activation Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Bulky glycocalyx drives cancer invasiveness by modulating substrate-specific adhesion

doi: 10.1101/2023.08.03.551677

Figure Lengend Snippet: (A) Representative confocal images of paxillin (red) and MUC1 (green) stained FACS sorted mucin-1 negative, intermediate (low) and high MCF7 cells in presence of DMSO control or 0.4 U/ml Neuraminidase and 20 µg/ml tunicamycin (NMase). Nuclei stained with DAPI. Bottom panel shows focal adhesion distribution with focal adhesion at the edge in white and adhesion inside in cyan. (B) Quantification of average number of total focal adhesion/cell, central focal adhesion/cell and average size of focal adhesions across different conditions (n=3, more than 50 cells per condition, ** p < 0.005, ns p > 0.05, data is presented as mean ± SEM). (C) Representative confocal images of phospho focal adhesion kinase (pFAK in green) and MUC1 (red) stained FACS sorted MCF7 cells and (D) Quantification of average number of pFAK focal adhesion/cell across different conditions (n=3, per condition ≥ 40 cells per condition, ** p < 0.005, ns p > 0.05, data is presented as mean ± SEM). (E) Focal adhesion dynamics in MDA-MB-231 cells in presence of DMSO control or 0.4 U/ml Neuraminidase and 20 µg/ml tunicamycin (NMase). Representative images showing focal adhesion at the beginning (0 min) and end (10 min) of experiment, and color-coded images depicting images of adhesions overlaid from multiple time points acquired over a period of 10 min. (F) Analysis of focal adhesion lifetime (*P<0.05; **P<0.001; NS p > 0.05, for n ≥ 16 cells analyzed per condition from three independent experiments, data is presented as mean ± SEM).

Article Snippet: Fixed cells were permeabilized in 0.1% Triton X-100 for 8 – 12 min, blocked using 5% BSA for 2 h at room temperature, and then incubated with primary antibodies (anti-MUC1 (CD227) Mouse monoclonal (BD, Cat# 555925), anti-paxillin rabbit monoclonal antibody (Abcam, Cat # ab32084),, anti phospho-focal adhesion kinase (pFAK) rabbit monoclonal antibody (Cat# sc-374668, Santa Cruz Biotechnology), anti MMP2 (Cat # 436000, Invitrogen) diluted in PBS overnight at 4°C.

Techniques: Staining, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Plasma gelsolin facilitates interaction between β 2 glycoprotein I and α 5 β 1 integrin

doi: 10.1111/j.1582-4934.2009.00940.x

Figure Lengend Snippet: Change in intracellular signalling by gelsolin and aCL/β 2 GPI. (A) Gelsolin and integrin affect phosphorylation of p38 MAPK by anti-β 2 GPI antibody. RAW264.7 cells were incubated for 2 hrs as indicated after serum-free culture for 16 hrs and then stimulated with aCL/β 2 GPI (WBCAL1) for 30 min., and then phosphorylation of p38 MAPK was determined by immunoblot analysis using specific antibodies against total-p38 and phospho-p38. (B) Anti-integrin α 5 β 1 antibody inhibits phosphorylation of FAK by aCL/β 2 GPI. RAW264.7 cells were stimulated with aCL/β 2 GPI for 10 min. and then phosphorylaion of FAK was determined by immunoblot analysis. (C) aCL/β 2 GPI increases NF-κB activity. RAW264.7 cells stably expressing kB luciferase reporter were inoculated into a 24-well dish and stimulated as indicated. After stimulation at 37°C for 4 hrs, kB luciferase activity was measured.

Article Snippet: The antibodies used in this study were as follows: mouse monoclonal anti-β 2 GPI antibody (WBCAL1; aCL/β 2 GPI [ ], and MAB1066, Chemicon International Inc., Temecula, CA, USA), mouse monoclonal anti-gelsolin antibody (clone 2, BD Transduction Laboratories, San Jose, CA, USA), rat monoclonal anti-integrin a 5 β 1 antibody (MAB1984, Chemicon International Inc.), mouse monoclonal anti-integrin β 1 antibody (Ha2/5, BD), rabbit polyclonal anti-p38 MAP kinase antibody (#9212, Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit polyclonal anti-phospho-p38 MAP kinase antibody (#9211, Cell Signaling Technology, Inc.), mouse monoclonal anti-focal adhesion kinase (FAK) antibody (clone 77, BD), mouse monoclonal anti-FAK(pY397)-phospho-specific antibody (clone 18, BD), mouse anti-b-actin (AC15, Sigma Chemical Co.) and mouse monoclonal anti-FLAG (M2) (Sigma Chemical Co.).

Techniques: Incubation, Western Blot, Activity Assay, Stable Transfection, Expressing, Luciferase