Journal: Journal of Cell Science

Article Title: Rnd3-induced cell rounding requires interaction with Plexin-B2

doi: 10.1242/jcs.192211

Figure Lengend Snippet: Rnd3 interacts with plexin-B2. (A) Schematic representation of plexin-B2 (full-length and cytoplasmic region) showing the extracellular Sema domain, plexins–semaphorins–integrins (PSI) domains and immunoglobulins–plexins–transcription factor (IPT) domains, and the intracellular split GAP domain, Rho-binding domain (RBD) and C-terminal PDZ-binding motif (PBM). (B,C) COS7 cells were transfected with expression vectors encoding VSV-epitope-tagged full-length plexin-B2 (B) or HA-epitope–plexin-B2-cyto (C). Cell lysates were incubated with GST or GST–Rnd3 on glutathione–Sepharose beads. Bound proteins were analysed by immunoblotting with the indicated antibodies. (D,E) Lysates of COS7 cells expressing full-length plexin-B2 (D) or HA–plexin-B2-cyto (E) and FLAG–Rnd3 were immunoprecipitated with anti-FLAG or anti-HA antibody, followed by immunoblotting. Full-length plexin-B2 migrates as a doublet at around 180 kDa. (F) Lysates of HeLa cells expressing FLAG–Rnd3 or transfected with empty vector (control) were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting for endogenous plexin-B2. Results from three independent experiments are shown in the blot. All other blots are representative of three independent experiments.

Article Snippet: Rabbit polyclonal anti-VSV-G antibody (V4888, Sigma-Aldrich) was used at 1:8000 for WB, mouse monoclonal Rnd3/RhoE antibody (hybridoma supernatant clone 4) ( ) at 1:5, mouse anti-GADPH antibody (MAB374, Millipore) at 1:10,000 and sheep anti-human plexin-B2 antibody (AF5329, R&D Systems) at 1:2000.

Techniques: Binding Assay, Transfection, Expressing, Incubation, Western Blot, Immunoprecipitation, Plasmid Preparation, Control

Journal: Journal of Cell Science

Article Title: Rnd3-induced cell rounding requires interaction with Plexin-B2

doi: 10.1242/jcs.192211

Figure Lengend Snippet: Comparison of Rnd protein interactions with plexin-B1, plexin-B2 and plexin-B3. (A) Alignment of RBD sequences from plexins B1, B2 and B3; * indicates identical amino acids, : indicates similar amino acids in all three sequences. (B) Lysates from COS7 cells expressing full-length VSV–plexin-B1, plexin-B2 or plexin-B3 were incubated with GST or GST–Rnd3 bound to glutathione–Sepharose beads. The bound proteins were analysed by immunoblotting with anti-VSV antibody. (C) Lysates of COS7 cells co-expressing VSV-tagged plexins B1, B2 or B3 and FLAG-tagged Rnd1, Rnd2 or Rnd3 were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-VSV antibody. (D) Relative levels of immunoprecipitated VSV–plexins (mean±s.e.m., n =3; ** P <0.01; **** P <0.0001); AU, arbitrary units. (E) Lysates of COS7 cells co-expressing HA–plexins B1, B2 or B3-cyto and FLAG-tagged Rnd1, Rnd2 or Rnd3 were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-FLAG antibody. (F) Lysates from COS7 cells expressing HA–plexin-B2-cyto and FLAG–Rnd3, FLAG–Rnd1 or FLAG–RhoA-G14V were immunoprecipitated with anti-HA antibody, followed by immunoblotting. All blots are representative of three independent experiments.

Article Snippet: Rabbit polyclonal anti-VSV-G antibody (V4888, Sigma-Aldrich) was used at 1:8000 for WB, mouse monoclonal Rnd3/RhoE antibody (hybridoma supernatant clone 4) ( ) at 1:5, mouse anti-GADPH antibody (MAB374, Millipore) at 1:10,000 and sheep anti-human plexin-B2 antibody (AF5329, R&D Systems) at 1:2000.

Techniques: Comparison, Expressing, Incubation, Western Blot, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: Rnd3-induced cell rounding requires interaction with Plexin-B2

doi: 10.1242/jcs.192211

Figure Lengend Snippet: Rap1A, Rap1B and R-Ras interact with plexin-B1 and plexin-B2. (A–C) COS7 cells were transfected with expression vectors encoding VSV-tagged full-length plexins B1 or B2 or B3 or HA-tagged plexin-B1-cyto or plexin-B2-cyto and (A) myc-R-Ras, (B) GFP–Rap1A or (C) HA–Rap1B. Cell lysates were immunoprecipitated (IP) and western blotted with the indicated antibodies. All blots are representative of three independent experiments. (D–G) COS7 cells were transfected with expression vectors encoding HA-tagged plexins B1, B2 or B3 (cytoplasmic regions) and/or FLAG-tagged Rnd1, Rnd2 or Rnd3 and (D) myc-R-Ras, (B) GFP–Rap1A or (F,G) GFP–RhoA. After 6 h, the medium was replaced with medium containing 1% FCS and left for 18 h. After 24 h, cells were lysed and GTPase activity levels analysed. The graphs show (D) R-Ras, (E) Rap1A or (F) RhoA activity levels normalized to the respective total protein, relative to control, or (G) total RhoA levels normalized to GADPH, relative to control. Values are mean±s.e.m. of three independent experiments; *** P <0.001; **** P <0.0001; ns, not significant; one-way ANOVA with Tukey posthoc test for multiple comparisons.

Article Snippet: Rabbit polyclonal anti-VSV-G antibody (V4888, Sigma-Aldrich) was used at 1:8000 for WB, mouse monoclonal Rnd3/RhoE antibody (hybridoma supernatant clone 4) ( ) at 1:5, mouse anti-GADPH antibody (MAB374, Millipore) at 1:10,000 and sheep anti-human plexin-B2 antibody (AF5329, R&D Systems) at 1:2000.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Activity Assay, Control

Journal: Journal of Cell Science

Article Title: Rnd3-induced cell rounding requires interaction with Plexin-B2

doi: 10.1242/jcs.192211

Figure Lengend Snippet: Rnd3 C-terminus and effector domains are required for plexin-B2 interaction. (A) Schematic representation of Rnd3 mutants used in this study. Full-length Rnd3 has N- and C-terminal extensions and a core GTP-binding region (residues 16–200). Rnd3ΔC contains amino acids 1–200, Rnd3ΔN amino acids 16–244 and Rnd3ΔNΔC amino acids 16–200. Point mutants are shown below. (B) Structure prediction analysis of the key residues involved in Rnd3 interaction with plexin-B RBD is represented as a ribbon diagram identifying Rnd3 effector domain and non-effector domain amino acids. Plexin-B1 RBD is shown in pink, Rnd1 in yellow (PDB 2REX), Rnd3 in cyan. (C–E) Lysates of COS7 cells expressing HA–plexin-B2-cyto and FLAG–Rnd1, FLAG–Rnd3 mutants or FLAG–RhoA-G14V were immunoprecipitated with anti-HA antibody followed by immunoblotting with the indicated antibodies. All blots are representative of three independent experiments; * indicates IgG or nonspecific bands.

Article Snippet: Rabbit polyclonal anti-VSV-G antibody (V4888, Sigma-Aldrich) was used at 1:8000 for WB, mouse monoclonal Rnd3/RhoE antibody (hybridoma supernatant clone 4) ( ) at 1:5, mouse anti-GADPH antibody (MAB374, Millipore) at 1:10,000 and sheep anti-human plexin-B2 antibody (AF5329, R&D Systems) at 1:2000.

Techniques: Binding Assay, Expressing, Immunoprecipitation, Western Blot

Journal: Journal of Cell Science

Article Title: Rnd3-induced cell rounding requires interaction with Plexin-B2

doi: 10.1242/jcs.192211

Figure Lengend Snippet: Rnd3 phosphorylation is not required for plexin-B2 interaction. (A,B) Lysates from COS7 cells expressing full-length VSV-tagged plexins B1, B2 or B3 (A) HA-tagged plexins B1, B2 or B3-cyto (B) and FLAG–Rnd3 or FLAG–Rnd3-AllA were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting. (C) COS7 cells expressing HA–plexin-B2-cyto, FLAG–Rnd3 and/or HA–14-3-3β were lysed and lysates immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with the indicated antibodies. All blots are representative of three independent experiments.

Article Snippet: Rabbit polyclonal anti-VSV-G antibody (V4888, Sigma-Aldrich) was used at 1:8000 for WB, mouse monoclonal Rnd3/RhoE antibody (hybridoma supernatant clone 4) ( ) at 1:5, mouse anti-GADPH antibody (MAB374, Millipore) at 1:10,000 and sheep anti-human plexin-B2 antibody (AF5329, R&D Systems) at 1:2000.

Techniques: Phospho-proteomics, Expressing, Immunoprecipitation, Western Blot

Journal: Journal of Cell Science

Article Title: Rnd3-induced cell rounding requires interaction with Plexin-B2

doi: 10.1242/jcs.192211

Figure Lengend Snippet: Plexin-B2 enhances Rnd3-induced cell rounding and inhibition of invasion. (A,B) HeLa cells were transfected with pCMV5 (empty vector control), FLAG–Rnd3 and/or full-length plexin-B2. After 16–18 h, cells were fixed and stained with anti-FLAG antibody and anti-plexin-B2 antibody. Actin filaments were visualized with AlexaFluor-546- or AlexaFluor-633-conjugated phalloidin. Scale bar: 10 µm. (B) Cell spread area (mean±s.e.m., n =3). Each dot represents a single cell; 19 cells were analysed for each condition. (C) HeLa cells were transfected with GFP (control), or GFP–Rnd3 with or without plexin-B2. Cell invasion was measured using Matrigel-coated Transwell filters. Invasion is shown relative to GFP-expressing control cells (mean±s.e.m., n =35). (D,E) HeLa cells (1×10 5 cells/ml) were transfected with pCMV5 (control), full-length plexin-B2 and wild-type (WT) FLAG–Rnd3 or FLAG–Rnd3 mutants that do not bind to plexin-B2 (Rnd3-V56Y, Rnd3-V87R and Rnd3-1–200). After 24 h, cells were fixed and stained for plexin-B2 (green) and FLAG–Rnd3 proteins (red). Actin filaments were detected with AlexaFluor-633-conjugated phalloidin (blue). Scale bar: 10 μm. (E) Mean percentage±s.e.m. of rounded cells from three independent experiments; n ≥100 cells per experiment. (F) HeLa cells were transfected with two siRNAs targeting plexin-B2 or with control siRNA. After 48 h, cells were transfected with FLAG-tagged Rnd3. Cells were lysed or fixed, permeabilized and immunostained 24 h later. Plexin-B2 depletion was determined by immunoblotting with anti-plexin-B2 antibody. Endogenous Rnd3 and exogenous Rnd3 were detected using anti-Rnd3 or anti-FLAG antibody, respectively. Blots are representative of three independent experiments. (G) Mean percentage±s.e.m. of rounded cells from three independent experiments; n ≥100 cells per experiment. For all graphs, * P <0.05, ** P <0.01, *** P <0.001 **** P <0.0001; ns, not significant; one-way ANOVA with Tukey posthoc test.

Article Snippet: Rabbit polyclonal anti-VSV-G antibody (V4888, Sigma-Aldrich) was used at 1:8000 for WB, mouse monoclonal Rnd3/RhoE antibody (hybridoma supernatant clone 4) ( ) at 1:5, mouse anti-GADPH antibody (MAB374, Millipore) at 1:10,000 and sheep anti-human plexin-B2 antibody (AF5329, R&D Systems) at 1:2000.

Techniques: Inhibition, Transfection, Plasmid Preparation, Control, Staining, Expressing, Western Blot