Journal: The Journal of Cell Biology

Article Title: Unc5B associates with LARG to mediate the action of repulsive guidance molecule

doi: 10.1083/jcb.200807029

Figure Lengend Snippet: Involvement of LARG in RhoA activation by RGMa. (A) Effects of knockdown of endogenous LARG expression using siRNA on RhoA activation by RGMa in transfected 293T cells. (B) The quantification of the data shown in A from six independent experiments. *, P < 0.01 compared with neogenin–VSV-G(+)/Unc5B-HA(+)/control siRNA(+)/LARG siRNA(−)/RGMa(−); **, P < 0.01 compared with (+)/(+)/(−)/(+)/(+). (C) Effects of myc–LARG PDZ transfection on RhoA activation induced by RGMa in neogenin–Unc5B-expressing 293T cells (left) and COS-7 cells (right). (D) Quantification of the data shown in C from three independent experiments. *, P < 0.05 compared with neogenin–VSV-G(+)/Unc5B-HA(+)/myc–LARG PDZ(−)/RGMa(−) or (+)/(+)/(+)/(+). (E) The RhoA activation assay in cortical neurons knocked down for LARG by siRNA. Western blots of the lysates panel show the knockdown of LARG expression by LARG siRNA but not by control siRNA. (F) Quantification of the data shown in E from four independent experiments. *, P < 0.05 compared with control siRNA(+)/LARG siRNA(−)/RGMa(−); **, P < 0.01 compared with (−)/(+)/(+). (G) LARG siRNA rescue experiment. Neurons were cotransfected with control siRNA and mock construct, LARG siRNA and mock construct, or LARG siRNA and myc-LARG construct. Western blots of the lysates panel show that knockdown of LARG expression by LARG siRNA was rescued by transfection of a plasmid encoding myc-LARG but not by mock plasmid. (H) The quantification of the data shown in G from three independent experiments. IB, immunoblotting; RBD, GST-rhotekin-Rho–binding domain. Error bars indicate SEM.

Article Snippet: After a brief centrifugation to remove the precleared beads, the cell lysates were incubated for 2 h (for co-IP with transfected 293T cell extracts) or overnight (for co-IP with rat cortical neuron extracts) at 4°C with anti–VSV-G antibody (Sigma-Aldrich), anti-HA antibody (Sigma-Aldrich), anti–c-myc antibody (Santa Cruz Biotechnology, Inc.), antineogenin (Santa Cruz Biotechnology, Inc.), anti-Unc5B (R&D Systems), or anti-LARG antibody (Santa Cruz Biotechnology, Inc.).

Techniques: Activation Assay, Knockdown, Expressing, Transfection, Control, Western Blot, Construct, Plasmid Preparation, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Unc5B associates with LARG to mediate the action of repulsive guidance molecule

doi: 10.1083/jcb.200807029

Figure Lengend Snippet: Interaction of Unc5B with LARG. (A and B) Co-IP of Unc5B with LARG in the transfected 293T cells. (C) Myc-LARGΔPDZ was not coimmunoprecipitated with Unc5B-HA in transfected 293T cells. The precipitated proteins with anti-myc antibody were immunoblotted with anti-HA or anti-myc antibody. (D) Co-IP of Unc5B-HA with myc–LARG PDZ in transfected 293T cells. (E and F) GST pull-down assay of GST–LARG PDZ with various deletion mutants of Unc5B ICD (E; ) and with Flag-tagged neogenin-ICD (F; neogenin-ICD-Flag). An anti-HA or Flag antibody was used for the detection of proteins bound to the beads. (G) Co-IP of Unc5B with LARG in rat cortical neurons. Lysates were immunoprecipitated with anti-Unc5B antibody (IP:Unc5B) or control IgG (IP:Control IgG). IB, immunoblotting.

Article Snippet: After a brief centrifugation to remove the precleared beads, the cell lysates were incubated for 2 h (for co-IP with transfected 293T cell extracts) or overnight (for co-IP with rat cortical neuron extracts) at 4°C with anti–VSV-G antibody (Sigma-Aldrich), anti-HA antibody (Sigma-Aldrich), anti–c-myc antibody (Santa Cruz Biotechnology, Inc.), antineogenin (Santa Cruz Biotechnology, Inc.), anti-Unc5B (R&D Systems), or anti-LARG antibody (Santa Cruz Biotechnology, Inc.).

Techniques: Co-Immunoprecipitation Assay, Transfection, Pull Down Assay, Immunoprecipitation, Control, Western Blot

Journal: British journal of pharmacology

Article Title: Regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signalling via interaction with Gli1.

doi: 10.1111/bph.15289

Figure Lengend Snippet: FIGURE 3 Wnt stimulates Smo-independent Gli1 nuclear translocation followed by LARG-mediated RhoA activation, leading to HSC contraction. LX2 human HSCs were treated with Wnt3a (50 ngml−1) and/or SIS3 (Smad3 inhibitor, 5 μM), SCH772984 (ERK inhibitor, 10 μM), MK2206 (AKT inhibitor, 5 μM), GANT-58 (Gli1 inhibitor, 5 μM), or PDTC (NF-κB inhibitor, 5 μM) for 24 h or transfected with β-catenin siRNA or Gli1 siRNA for 48 h. (a) Western blotting of phospho-MLC2; n = 5. (b) Collagen gel contraction; n = 5. (c) Cytoskeleton immunofluorescence (400× magnification, scale bars: 10 μm); n = 3. (d) Western blotting of active and total RhoA; n = 5. (e) Western blotting of LARG in whole cell lysates; n = 5. (f) Western blotting of active LARG in membrane lysates; n = 5. *P < .05, significantly different from vehicle control or control siRNA, #P < .05, significantly different from Wnt3a or Wnt3a + control siRNA, N.S., no significance

Article Snippet: Rabbit or mouse polyclonal antibodies against RhoA (10749-1-AP, for WB 1:1,000 dilution), LARG (22441-1-AP, for WB 1:1,000 dilution), and β-catenin (17565-1-AP, for IHC 1:200 dilution, and IF 1:200 dilution) were purchased from Proteintech Group (Chicago, IL, USA).

Techniques: Translocation Assay, Activation Assay, Transfection, Western Blot, Immunofluorescence, Membrane, Control

Journal: British journal of pharmacology

Article Title: Regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signalling via interaction with Gli1.

doi: 10.1111/bph.15289

Figure Lengend Snippet: FIGURE 9 Scheme of the molecular mechanisms underlying HSC contraction. Activation of Wnt/β-catenin signalling represses transcription of Sufu in a TCF4-dependent manner. This stimulates Gli1 nuclear translocation, leading to LARG-associated RhoA activation and results in contraction of HSCs. β-Catenin inhibitors reduce HSC contraction by disrupting this cascade and thereby exhibit therapeutic effects in models of cirrhotic portal hypertension

Article Snippet: Rabbit or mouse polyclonal antibodies against RhoA (10749-1-AP, for WB 1:1,000 dilution), LARG (22441-1-AP, for WB 1:1,000 dilution), and β-catenin (17565-1-AP, for IHC 1:200 dilution, and IF 1:200 dilution) were purchased from Proteintech Group (Chicago, IL, USA).

Techniques: Activation Assay, Translocation Assay