Journal: Cell adhesion & migration

Article Title: Adhesion structures in leukemia cells and their regulation by Src family kinases.

doi: 10.1080/19336918.2017.1344796

Figure Lengend Snippet: Figure 4. Effect of SFK inhibitors on Lyn activity. OCI-AML3 cells were treated with dasatinib or SKI-1, either in suspension (susp) or after adhesion to FN-coated surface (FN). Lyn activity was assessed from the extent of phosphorylation at Lyn autophos- phorylation site (Tyr397) and on 2 known Lyn substrates, HS-1 and Pyk2. Lyn autophosphorylation was analyzed from bands at 53 and 56 kDa on western-blots incubated with pan anti-pSFK antibody. A-B: Representative western-blots from cells treated for 1 h with increasing concentrations of dasatinib (A) or SKI-1 (B). C: Summary results from 4 experiments performed with 20 mM SKI- 1 or 100 nM dasatinib treatment of pre-adhered OCI-AML3 cells (cell binding to FN for 1.5 h, then 30 min inhibitor treatment). Note that the exposition time for different membranes was not the same and was optimized to show the changes in pSFK level under different conditions.

Article Snippet: Antibodies against the following targets were used: phospho-Src Family (Tyr416) (Cell Signaling, #2101), c-Src (Calbiochem, #OP07), Lyn (BD Biosciences, #610003), Hck (BD Biosciences, #610278), Lck (Abcam, #ab 32149), vinculin (Abcam, #ab129002), paxillin (BD Biosciences, #610051), talin 1 (Abcam, #ab157808), phospho-HS-1 (Tyr397) (Cell Signaling, #8714S), phospho-Pyk2 (Tyr402) (Life Technologies, #44–618G), b-actin (Sigma, #A5441).

Techniques: Activity Assay, Suspension, Phospho-proteomics, Western Blot, Incubation, Binding Assay

Journal: Frontiers in Aging Neuroscience

Article Title: CCL5 promotes LFA-1 expression in Th17 cells and induces LCK and ZAP70 activation in a mouse model of Parkinson’s disease

doi: 10.3389/fnagi.2023.1250685

Figure Lengend Snippet: CCL5 promoted Th17 cell differentiation and LFA-1 expression by activating LCK and ZAP70. (A) Flow cytometry and FlowJo software were used to analyze peripheral PBMCs from mice. The proportions of IL-17A + , IL-17A + and LFA-1 + , IL-17A + and pLCK + , IL-17A + and pZAP70 + cells among CD4 + cell populations were calculated n = 5 per group. (B) Naive CD4 + T cells that were cultured in vitro were induced to differentiate into Th17 cells, which were treated with CCL5 or PBS. After 3 days, LFA-1, pLCK, pZAP70, total LCK, total ZAP70 and Rorγt levels were measured by Western blot analysis and quantified using ImageJ n = 3 per group. Data are presented as the means ± SEMs. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The cells were then resuspended in fixation and permeabilization buffer for 45 min. After 45 min of incubation in the dark, the cells were incubated with a diluted PE-labeled antibody targeting the Th17-specific marker IL-17A (5 μg/mL, BD Biosciences, 559502), PE-labeled antibody targeting pLCK (1:50, Cell Signaling Technology, 14791S), PE-labeled antibody targeting pZAP70 (1:50, Cell Signaling Technology, 94361S), PEcy7-labeled antibody targeting IFN-gamma (5 μg/mL, Invitrogen, 2153357) or PEcy7-labeled antibody targeting Foxp3 (5 μg/mL, Invitrogen, 2378858) at 4°C in the dark for another 45 min. A BV421-labeled antibody targeting the Th17-specific marker IL-17A (5 μg/mL, BioLegend, 506925) was used to perform pZAP70 or pLCK staining.

Techniques: Cell Differentiation, Expressing, Flow Cytometry, Software, Cell Culture, In Vitro, Western Blot

Journal: Frontiers in Aging Neuroscience

Article Title: CCL5 promotes LFA-1 expression in Th17 cells and induces LCK and ZAP70 activation in a mouse model of Parkinson’s disease

doi: 10.3389/fnagi.2023.1250685

Figure Lengend Snippet: PP2 improved motor symptoms in mice, inhibited Th17 cell differentiation and reduced LFA-1 expression by inhibiting LCK and ZAP70 activation. (A) The timeline of the mouse treatment regimen. (B) Rotarod test. (C) Pole test. (D–H) After PP2 treatment, flow cytometry and FlowJo software were used to analyze peripheral PBMCs from the mice. The proportions of IL-17A + , IL-17A + and CCR5 + , IL-17A + and LFA-1 + , IL-17A + and pLCK + , IL-17A + and pZAP70 + cells among CD4 + cell populations were calculated n = 5 per group. Data are presented as the means ± SEMs. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The cells were then resuspended in fixation and permeabilization buffer for 45 min. After 45 min of incubation in the dark, the cells were incubated with a diluted PE-labeled antibody targeting the Th17-specific marker IL-17A (5 μg/mL, BD Biosciences, 559502), PE-labeled antibody targeting pLCK (1:50, Cell Signaling Technology, 14791S), PE-labeled antibody targeting pZAP70 (1:50, Cell Signaling Technology, 94361S), PEcy7-labeled antibody targeting IFN-gamma (5 μg/mL, Invitrogen, 2153357) or PEcy7-labeled antibody targeting Foxp3 (5 μg/mL, Invitrogen, 2378858) at 4°C in the dark for another 45 min. A BV421-labeled antibody targeting the Th17-specific marker IL-17A (5 μg/mL, BioLegend, 506925) was used to perform pZAP70 or pLCK staining.

Techniques: Cell Differentiation, Expressing, Activation Assay, Flow Cytometry, Software

Journal: Frontiers in Aging Neuroscience

Article Title: CCL5 promotes LFA-1 expression in Th17 cells and induces LCK and ZAP70 activation in a mouse model of Parkinson’s disease

doi: 10.3389/fnagi.2023.1250685

Figure Lengend Snippet: PP2 inhibited Th17 cell differentiation and LFA-1 expression in vitro . Naive CD4 + T cells from the spleens of C57BL/6J mice were purified by magnetic beads in vitro and induced to differentiate into Th17 cells. CCL5 and PP2 were used for treatment. After 3 days, flow cytometry and FlowJo software were used to analyze the cells. The proportions of IL-17A + , IL-17A + and CCR5 + , IL-17A + and LFA-1 + , IL-17A + and pLCK + , and IL-17A + and pZAP70 + cells among CD4 + cell populations were calculated n = 3 per group. Data are presented as the means ± SEMs. * p < 0.05 and ** p < 0.01.

Article Snippet: The cells were then resuspended in fixation and permeabilization buffer for 45 min. After 45 min of incubation in the dark, the cells were incubated with a diluted PE-labeled antibody targeting the Th17-specific marker IL-17A (5 μg/mL, BD Biosciences, 559502), PE-labeled antibody targeting pLCK (1:50, Cell Signaling Technology, 14791S), PE-labeled antibody targeting pZAP70 (1:50, Cell Signaling Technology, 94361S), PEcy7-labeled antibody targeting IFN-gamma (5 μg/mL, Invitrogen, 2153357) or PEcy7-labeled antibody targeting Foxp3 (5 μg/mL, Invitrogen, 2378858) at 4°C in the dark for another 45 min. A BV421-labeled antibody targeting the Th17-specific marker IL-17A (5 μg/mL, BioLegend, 506925) was used to perform pZAP70 or pLCK staining.

Techniques: Cell Differentiation, Expressing, In Vitro, Purification, Magnetic Beads, Flow Cytometry, Software