Journal: Fundamental Research

Article Title: TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

doi: 10.1016/j.fmre.2022.01.017

Figure Lengend Snippet: TRPC5 regulates contractions via cytosolic phospholipase A 2 (cPLA 2 ) in the high-fat diet (HFD)-induced obese mouse aorta. (a) Western blots and analysis of cPLA 2 and phosphorylated cPLA 2 (p-cPLA 2 ) expression in normal-fat diet (NFD, n = 15), AM237 (100 nmol/L)-treated NFD ( n = 9), HFD-induced obese ( n = 14), clemizole (20 μmol/L)-treated HFD ( n = 7), and TRPC5 −/− -HFD ( n = 5) mouse aortic endothelial cells (MAoECs). (b) Dose-dependent effect of AM237 on p-cPLA 2 levels in NFD MAoECs. AM237 (nmol/L), 0, n = 16; 50, n = 11; 100, n = 16; 200, n = 10. (c) Dose-dependent effect of clemizole on p-cPLA 2 levels in HFD MAoECs ( n = 5). (d) Representative fluorescence images of the Bis-BODIPY™ FL C 11 -PC stained en-face aorta and analysis of PLA 2 activity in endothelial cells of the NFD ( n = 9), AM237 (100 nmol/L)-pretreated NFD ( n = 7), HFD ( n = 17), clemizole (20 μmol/L)-treated HFD ( n = 9), and TRPC5 −/− HFD ( n = 12) mouse aorta (scale bars, 10 μm). (e) Acetylcholine (ACh)-induced contraction in HFD ( n = 4) and MAFP (10 μmol/L)-treated HFD ( n = 6) mouse aortic rings. (f) ACh-induced contraction in the NFD ( n = 5), AM237 (100 nmol/L)-pretreated NFD ( n = 5), and AM237 (100 nmol/L) +MAFP (30 μmol/L)-pretreated NFD ( n = 6) mouse aorta. Mean ± SEM; a, * P < 0.05 vs NFD, # P < 0.05 vs HFD, NS, no significant difference, Kruskal-Wallis and Dunn's post hoc non-parametric test (p-cPLA 2 ) and one-way ANOVA followed by Turkey's multiple comparisons test (cPLA 2 ); b, * P < 0.05, NS, no significant difference vs no AM237, one-way ANOVA followed by Dunnett's multiple comparisons test; c, * P < 0.05, NS, no significant difference vs no clemizole, one-way ANOVA followed by Dunnett's multiple comparisons test; d, * P < 0.05 vs NFD, # P < 0.05 vs HFD, one-way ANOVA followed by Turkey's multiple comparisons test; e, * P < 0.05 vs CTL, two-way ANOVA followed by Bonferroni test (left) and Student's unpaired two-tailed t test (right); f, * P < 0.05 vs CTL, # P < 0.05 vs AM237, two-way ANOVA followed by Bonferroni test (left) and one-way ANOVA followed by Turkey's multiple comparisons test (right).

Article Snippet: The membranes were incubated overnight at 4 °C with the primary antibodies anti-TRPC5 (1:200, Proteintech), anti-COX-1 (1:200, Abcam), anti-COX-2 (1:2000, Abcam), anti-cPLA 2 (1:200, Santa Cruz), anti-p-cPLA 2 (1:1000, Signalway Antibody), and anti-GAPDH (1:1000, Santa Cruz) followed by horseradish peroxidase-conjugated secondary antibody (mouse, 1:10,000; rabbit, 1:5000, Beyotime) at room temperature for 2 h. ImageJ was used for band intensity analysis.

Techniques: Western Blot, Expressing, Fluorescence, Staining, Activity Assay, Two Tailed Test

Journal: eNeuro

Article Title: Cyclin-Dependent Kinase 5 Regulates cPLA2 Activity and Neuroinflammation in Parkinson’s Disease

doi: 10.1523/ENEURO.0180-22.2022

Figure Lengend Snippet: The expression of cPLA2 wt and its mutants in the membrane and cytosolic protein fraction. A , The expression pattern of cPLA2 and its mutants in a membrane and cytosolic fraction of HEK293T protein lysate cotransfected with cPLA2 and its mutants along with Cdk5/p35 and Cdk5/p25. The reaction standard used for membrane fraction is calnexin, and for cytosolic fraction is β-tubulin. B , Cytosolic and membrane fraction fold changes over control. C , Membrane translocation of phosS268 cPLA2 in the presence of active Cdk5 kinase. D , Membrane translocation of phospho-T505 cPLA2 in the presence of active Cdk5 Kinase. E , Densitometry of relative expressions of p-cPLA2-268 in cytosolic and membrane fractions. F , Densitometry of p-cPLA2-505 in cytosolic and membrane fractions. Calnexin is used as membrane fraction control, and β-tubulin as cytosolic frantic control. Data are presented as the mean ± SEM, and analysis was performed using a one-way ANOVA followed by Bonferroni’s post hoc test; n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies used in the Western blot experiment are 6×-His tag (1 μg/ml; catalog #NBP2-61482, Novus Biologicals) for cPLA2 variants, Calnexin (1:5000; stock dilution, 1 mg/ml; catalog #SAB4503258, Sigma-Aldrich), cPLA2 (1:1000; stock dilution, 1.5 mg/ml; catalog #SAB4200211, Sigma-Aldrich), p-cPLA2-Ser-505 (1:1000; stock dilution, 1 mg/ml; catalog #SAB4503812, Sigma-Aldrich), Cdk5 (1:1000; stock dilution, 5 μg/ml; catalog #AHZ0492, Thermo Fisher Scientific), p35/25 (1:5000; stock dilution, 1 mg/ml; catalog #C64B10, Cell Signaling Technology), p-cPLA2-Thr-268 (1:500; GenScript Biotech), and β-tubulin (1:10,000; stock dilution, 1 mg/ml; catalog #T8328, Sigma-Aldrich).

Techniques: Expressing, Membrane, Control, Translocation Assay

Journal: eNeuro

Article Title: Cyclin-Dependent Kinase 5 Regulates cPLA2 Activity and Neuroinflammation in Parkinson’s Disease

doi: 10.1523/ENEURO.0180-22.2022

Figure Lengend Snippet: Comparison of wild-type and mutant structural forms using MD simulations. A , Representative snapshots of MD simulations derived structure at the initial (0 ns) and last (1 μs) of wild and mutant systems. The protein structures are colored according to cPLA2 domain architecture, where C2 domain is highlighted in blue, linker in magenta, and catalytic domain (KD) in orange. B , Angular distribution between domains in wild and mutant systems during the last 150 ns of the trajectory. C , Secondary structure evolution of the catalytic domain residues (144–49) according to DSSP analysis in wild and mutant trajectories. D , Intramolecular network near the mutant sites for wild and double mutant systems at 1 μs. The size of the nodes represents the degree. The edges are represented as types of interaction, where hydrogen bonds are highlighted in blue, and contacts in gray. E , Snapshots highlighting these sites in ball and stick representation and surface hydrophobicity shown in blue and gold.

Article Snippet: The antibodies used in the Western blot experiment are 6×-His tag (1 μg/ml; catalog #NBP2-61482, Novus Biologicals) for cPLA2 variants, Calnexin (1:5000; stock dilution, 1 mg/ml; catalog #SAB4503258, Sigma-Aldrich), cPLA2 (1:1000; stock dilution, 1.5 mg/ml; catalog #SAB4200211, Sigma-Aldrich), p-cPLA2-Ser-505 (1:1000; stock dilution, 1 mg/ml; catalog #SAB4503812, Sigma-Aldrich), Cdk5 (1:1000; stock dilution, 5 μg/ml; catalog #AHZ0492, Thermo Fisher Scientific), p35/25 (1:5000; stock dilution, 1 mg/ml; catalog #C64B10, Cell Signaling Technology), p-cPLA2-Thr-268 (1:500; GenScript Biotech), and β-tubulin (1:10,000; stock dilution, 1 mg/ml; catalog #T8328, Sigma-Aldrich).

Techniques: Comparison, Mutagenesis, Derivative Assay

Journal: eNeuro

Article Title: Cyclin-Dependent Kinase 5 Regulates cPLA2 Activity and Neuroinflammation in Parkinson’s Disease

doi: 10.1523/ENEURO.0180-22.2022

Figure Lengend Snippet: Cdk5 expression, generation of p25, its kinase activity, and release of arachidonic acid and prostaglandin E2 synthesis by astrocytes exposed to MPP + and TFP5 treatment. A , The image shows a blot with the expression of Cdk5 in control versus MPP + (10 μ m ) along with MPP + (10 μ m ) versus MPP + (10 μ m ) plus TFP5 and SCP treatment in the protein lysate of astrocytes. B , Bar diagram showing densitometric analysis of Cdk5 expression over β-actin used as a standard for control, MPP + (10 μ m ), MPP + (10 μ m ) + TFP5 and MPP + (10 μ m ) + SCP, respectively. C , The bar diagram represents Cdk5 activity in the astrocyte protein lysate with control versus MPP + (10 μ m ) and MPP + (10 μ m ) versus MPP + (10 μ m ) + TFP5. D , The expression pattern of p35 and p25 in control, MPP + (10 μ m ), MPP + (10 μ m ) + TFP5, and MPP + (10 μ m ) + SCP, respectively. β-Actin was used as a standard. E , The blot representing the expression pattern of cPLA2 in control, MPP + (10 μ m ), MPP + (10 μ m ) + TFP5, and MPP + (10 μ m ) + SCP, respectively. F , The densitometric graph showing cPLA2 expression over β-actin of the above blot. G , The bar graph showing fold changes in cPLA2 activity in astrocyte lysate of control versus MPP + (10 μ m ) and MPP + (10 μ m ) versus MPP + (10 μ m ) + TFP5. H , The scatter plot showing 3H-arachidonic acid release from control astrocytes along with MPP + (10 μ m ), MPP + (10 μ m ) + TFP5, and MPP + (10 μ m ) + SCP. I , The bar graph showing prostaglandin E2 synthesis in control versus MPP + (10 μ m ) and MPP + (10 μ m ) versus MPP + (10 μ m ) + TFP5 along with MPP + (10 μ m ) + SCP as the standard for inhibitor treatment. Data are presented as the mean ± SEM, and analysis was performed using one-way ANOVA and Bonferroni’s test; n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, not significant (ns).

Article Snippet: The antibodies used in the Western blot experiment are 6×-His tag (1 μg/ml; catalog #NBP2-61482, Novus Biologicals) for cPLA2 variants, Calnexin (1:5000; stock dilution, 1 mg/ml; catalog #SAB4503258, Sigma-Aldrich), cPLA2 (1:1000; stock dilution, 1.5 mg/ml; catalog #SAB4200211, Sigma-Aldrich), p-cPLA2-Ser-505 (1:1000; stock dilution, 1 mg/ml; catalog #SAB4503812, Sigma-Aldrich), Cdk5 (1:1000; stock dilution, 5 μg/ml; catalog #AHZ0492, Thermo Fisher Scientific), p35/25 (1:5000; stock dilution, 1 mg/ml; catalog #C64B10, Cell Signaling Technology), p-cPLA2-Thr-268 (1:500; GenScript Biotech), and β-tubulin (1:10,000; stock dilution, 1 mg/ml; catalog #T8328, Sigma-Aldrich).

Techniques: Expressing, Activity Assay, Control

Journal: eNeuro

Article Title: Cyclin-Dependent Kinase 5 Regulates cPLA2 Activity and Neuroinflammation in Parkinson’s Disease

doi: 10.1523/ENEURO.0180-22.2022

Figure Lengend Snippet: cPLA2 expression, its activity, and prostaglandin E2 synthesis in neuroglia cells with MPP + and TFP5 treatment. A , The blot showing cPLA2 expression pattern of control, MPP + (10 μ m ), MPP + (10 μ m ) + TFP5 and MPP + (10 μ m ) + SCP in protein lysate of neuronal–glial culture along with its densitometric analysis. B , The bar graph for the fold of change in cPLA2 activity of control versus MPP + (10 μ m ) and MPP + (10 μ m ) versus MPP + (10 μ m ) + TFP5 in neuronal–glial culture. C , The bar graph shows prostaglandin E2 amount in control versus MPP + (10 μ m ) and MPP + (10 μ m ) versus MPP + (10 μ m ) + TFP5 in neuronal–glial culture. The MPP + (10 μ m ) + SCP is used as a standard reaction for inhibitor treatment; n = 3. Data are presented as the mean ± SEM, and analysis was done using one-way ANOVA and Bonferroni’s test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies used in the Western blot experiment are 6×-His tag (1 μg/ml; catalog #NBP2-61482, Novus Biologicals) for cPLA2 variants, Calnexin (1:5000; stock dilution, 1 mg/ml; catalog #SAB4503258, Sigma-Aldrich), cPLA2 (1:1000; stock dilution, 1.5 mg/ml; catalog #SAB4200211, Sigma-Aldrich), p-cPLA2-Ser-505 (1:1000; stock dilution, 1 mg/ml; catalog #SAB4503812, Sigma-Aldrich), Cdk5 (1:1000; stock dilution, 5 μg/ml; catalog #AHZ0492, Thermo Fisher Scientific), p35/25 (1:5000; stock dilution, 1 mg/ml; catalog #C64B10, Cell Signaling Technology), p-cPLA2-Thr-268 (1:500; GenScript Biotech), and β-tubulin (1:10,000; stock dilution, 1 mg/ml; catalog #T8328, Sigma-Aldrich).

Techniques: Expressing, Activity Assay, Control