Journal: PLoS ONE

Article Title: Enhanced Neurite Outgrowth of Human Model (NT2) Neurons by Small-Molecule Inhibitors of Rho/ROCK Signaling

doi: 10.1371/journal.pone.0118536

Figure Lengend Snippet: (A) Measurements of neurite outgrowth after 24 h incubation with elevated levels of the Rho activator lysophosphatidic acid (LPA) showed no change in neurite lengths, whereas Ibuprofen increased neurite length (2 independent experiments, 470 to 947 neurites measured). (B) Within 45 min, neurons cultured under control conditions showed a slowly advancing growth cone. (C) Inhibition of RhoA using 500 μM Ibuprofen elicited a weak positive effect on filopodial formation and extension of growth cone. (D) RhoA activation using 100 μM LPA resulted in a growth cone collapse and retraction of the neurite. ***p<0.001 against control by Kruskal-Wallis. Scale bar is 25 μm.

Article Snippet: The RhoA Activation Assay Biochem Kit (bead pull-down format) was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Incubation, Cell Culture, Control, Inhibition, Activation Assay

Journal: PLoS ONE

Article Title: Enhanced Neurite Outgrowth of Human Model (NT2) Neurons by Small-Molecule Inhibitors of Rho/ROCK Signaling

doi: 10.1371/journal.pone.0118536

Figure Lengend Snippet: (A) A RhoA bead pull down assay revealed lowered levels of activated RhoA in Ibuprofen treated cells. Treatment with downstream Rho kinase inhibitor Y-27632 showed no difference in RhoA level compared to control. After treatment with lysophosphatidic acid, significantly more phosphorylated RhoA could be detected in the Western Blot. Treatment with downstream Rho kinase inhibitor Fasudil showed no difference in RhoA level compared to control. Cell lysates were blotted and stained with RhoA-specific monoclonal antibody. Staining is visualized with horseradish peroxidase and the 3, 3′-diaminobenzidine substrate. Molecular weight of stained protein bands corresponds to about 20 kDa as reported for RhoA. (B) Relative RhoA level was decreased to approximately 50% of control after Ibuprofen treatment whereas RhoA level after LPA treatment was increased to 200% of control. Data are normalized grey values ± SEM of 4 independent experiments.

Article Snippet: The RhoA Activation Assay Biochem Kit (bead pull-down format) was purchased from Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Pull Down Assay, Control, Western Blot, Staining, Molecular Weight

Journal: PLoS ONE

Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

doi: 10.1371/journal.pone.0112753

Figure Lengend Snippet: A) Effects of CD148 in E-cadherin contact formation were examined by a calcium-switch assay and immunofluorescence staining. CD148 WT, but not CS, promotes E-cadherin contact formation in A431D/E-cadherin WT cells, as evidenced by the expanded and intense E-cadherin distribution (left panels), while CD148 WT shows no effects in A431D/E-cadherin 764 AAA cells (right panels). B) Activities of Rac1, Cdc42, and RhoA were measured in the condition of calcium switch assay. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by the pull-down assays and/or immunoblotting described in the “ ” (left panels). The activity was normalized to total amount of protein using densitometry and expressed as fold change (right panels). The data show means ± SEM of quadruplicate determinations. **P<0.05 vs. CD148 (−) cells.

Article Snippet: RhoA, Rac1, and Cdc42 activities were assessed using the Rac1 and RhoA pull-down activation assay kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer’s instruction.

Techniques: Immunofluorescence, Staining, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: CD148 Tyrosine Phosphatase Promotes Cadherin Cell Adhesion

doi: 10.1371/journal.pone.0112753

Figure Lengend Snippet: A and B) CD148-introduced or CD148-negative A431D/E-cadherin WT (panel A) and A431D/E-cadherin 764 AAA (panel B) cells were subjected to a hanging drop assay. Rac1, Cdc42, and RhoA activities were assessed at the indicated time points. Active and total levels of Rac1, Cdc42, and RhoA proteins were assessed by pull-down assays and/or immunoblot analysis (left panels). The relative levels of active versus total Rac1, Cdc42, and RhoA were quantified by densitometric analysis (right panels). The data show means ± SEM of quadruplicate determinations. **P<0.05 vs. CD148 (−) cells. CD148 WT, but not CS, increases Rac1 activity in A431D/E-cadherin WT cells, while this effect is not observed in A431D/E-cadherin 764 AAA cells. C) Effects of CD148 WT in cell-cell adhesion were assessed by a hanging drop assay in the presence or absence of Rac1 inhibitor NSC 23766 (75 µM). CD148 increase of cell-cell adhesion is largely diminished by Rac1 inhibition.

Article Snippet: RhoA, Rac1, and Cdc42 activities were assessed using the Rac1 and RhoA pull-down activation assay kit (Cytoskeleton, Inc., Denver, CO) according to the manufacturer’s instruction.

Techniques: Western Blot, Activity Assay, Inhibition