Journal: PLoS ONE

Article Title: Modulation of Phosducin-Like Protein 3 (PhLP3) Levels Promotes Cytoskeletal Remodelling in a MAPK and RhoA-Dependent Manner

doi: 10.1371/journal.pone.0028271

Figure Lengend Snippet: A commercially available RhoA-GTP/Rhotekin pull down assay was carried out on siRNA silenced PhLP3 LB01 cells. Samples were collected 24 h, 48 h and 72 h post transfection and activated RhoA was detected at 72 h post-knockdown with an anti-Rho A antibody but not in plasmid only control cells (A). LB01 cells transfected as in panel A for 72 h were then treated for 24 h with the ROCK inhibitor Y2763 (either 3 µM or 10 µM), fixed and stained for actin (rhodamine phalloidin, red). F-actin disassembly was observed at both Y2763 concentrations and the concentration of peripheral actin was observed with 10 µM Y2763 (B).

Article Snippet: Activation of RhoA was determined using a commercially available pull down assay based upon detecting the interaction of the active GTP-bound RhoA with the Rho-binding domain (RBD) of Rhotekin (RhoA Activation Assay Kit, STA-403-A, Cell Biolabs Inc, USA).

Techniques: Pull Down Assay, Transfection, Knockdown, Plasmid Preparation, Control, Staining, Concentration Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: LINC00891 regulated by miR-128-3p/GATA2 axis impedes lung cancer cell proliferation, invasion and EMT by inhibiting RhoA pathway

doi: 10.3724/abbs.2022005

Figure Lengend Snippet: LINC00891 regulates A549 and H460 cell functions via the RhoA pathway (A) A549 and H460 cells were transfected with LINC00891 overexpression vector, LINC00891 siRNA or respective negative controls, and the activated RhoA was determined using RhoA Activation Assay Kit. (B) Cells were treated with 15 μM CCG-1423 for 24 h, and the activated RhoA was assessed using RhoA Activation Assay Kit. Cells were treated with LINC00891 siRNA alone or together with 15 μM CCG-1423, and cell proliferation (C), invasion (D), and migration (E) were analyzed. (F) Western blot analysis was used to measure the protein levels of E-cadherin, Vimentin, Snail and Slug. n=5 in each group, *P<0.05, **P<0.01.

Article Snippet: RhoA activity was assessed using RhoA Activation Assay Kit (Merck Millipore, Billerica, USA) according to the manufacturer’s instructions.

Techniques: Transfection, Over Expression, Plasmid Preparation, Activation Assay, Migration, Western Blot

Journal: JCI Insight

Article Title: EGFR inhibition leads to enhanced desmosome assembly and cardiomyocyte cohesion via ROCK activation

doi: 10.1172/jci.insight.163763

Figure Lengend Snippet: ( A ) RhoA G-LISA showing enhanced RhoA activity upon EGFR inhibition by erlotinib. * P ≤ 0.05, unpaired Student’s t test, n = 3. ( B ) Representative Western blot showing phosphorylation of the ROCK target MLC2 upon erlotinib treatment. * P ≤ 0.05, 2-way ANOVA with Holm Sidak’s multiple comparison test, n = 6 independent experiments. ( C ) Dispase-based dissociation assay in HL-1 cardiomyocytes, after treatment with erlotinib with and without Y27632. Y27632 was added 30 minutes prior to erlotinib incubation for 60 minutes, with representative pictures of the wells. * P ≤ 0.05, 2-way ANOVA with Holm Sidak’s multiple comparison test, n = 8 independent experiments. ( D ) Immunostaining of DP and DSG2 in HL-1 cardiomyocytes with WGA as membrane marker after erlotinib treatment with and without Y26732, as in C . White arrows indicate areas of increased DP or DSG2 recruitment to the cell membrane. Scale bar: 10 μm. ( E ) Dispase-based dissociation assay in HL-1 cardiomyocytes after 90 minutes of Ca 2+ depletion and treatment with erlotinib with and without Y26732 with representative pictures of the wells. * P ≤ 0.05, 2-way ANOVA with Holm-Sidak’s multiple comparison test, n = 7 independent experiments.

Article Snippet: RhoA G-LISA was performed using the RhoA G-LISA Activation Assay kit (Biozol, BK124), which measures cellular levels of GTP-loaded RhoA, according to the manufacturer’s instructions.

Techniques: Activity Assay, Inhibition, Western Blot, Phospho-proteomics, Comparison, Incubation, Immunostaining, Membrane, Marker

Journal: Neural Regeneration Research

Article Title: Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells

doi: 10.4103/1673-5374.239438

Figure Lengend Snippet: Effects of exogenous MAG treatment on RhoA activity in neuro-2a cells (ELISA assay). Data are expressed as the mean ± SE ( n = 6 per group; one-way analysis of variance followed by t -test). ** P < 0.01, *** P < 0.001. ELISA: Enzyme-linked immunosorbent assay; MAG: myelin-associated glycoprotein; ns: not significant; min: minutes.

Article Snippet: The activity of RhoA was measured using a commercial RhoA activity assay ELISA kit (Jingma Bioscientifics, Inc., Shanghai, China).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay