Journal: PLoS ONE

Article Title: The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA

doi: 10.1371/journal.pone.0105661

Figure Lengend Snippet: ( A, B ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (PRG) alone or together with kinase-deficient mutants of HA-tagged IKKα (K44M) (IKKα-KD) or HA-tagged IKKβ (K44M) (IKKβ-KD) including SRE.L reporter (RepLucdelCMV) (A) or NF-kB-dependent luciferase reporter plasmid (NF-κB-Luc) (B). 48 hours after transfection, cells were incubated with 25 ng/ml TNFα or 150 nM Sema4D for 8 hours (as indicated), and luciferase activity was determined. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05. ( C, D ) MCF-7 cells were treated with SC-514 (50 µM) or NBDBP (100 µM) for the indicated time periods. After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes (C) or 10 ng/ml EGF for 20 minutes (D), cells were lysed, and a specific antibody directed against the phosphorylated version of ErbB-2 was used to visualize ErbB-2 phosphorylation. ErbB-2 levels in lysed samples were controlled using an anti-ErbB-2 antibody. Shown are representative examples of at least three experiments.

Article Snippet: For precipitation of endogenous Plexin-B1 in MCF-7 cells, we used an anti-Plexin-B1 antibody (R&D Systems).

Techniques: Transfection, Luciferase, Plasmid Preparation, Incubation, Activity Assay, Phospho-proteomics

Journal: PLoS ONE

Article Title: The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA

doi: 10.1371/journal.pone.0105661

Figure Lengend Snippet: ( A ) After incubation with Sema4D (150 nM) or TNFα (25 ng/ml) for the indicated time periods, MCF-7 cells were lysed, and IκBα degradation was visualized using an anti-IκBα antibody. ( B ) MCF-7 cells were treated with TNFα (25 ng/ml), Sema4D (150 nM) or control buffer (PBS) for 20 minutes and lysed. IKKα/β proteins were precipitated using an anti-IKKα/β antibody. Precipitates were further processed and subjected to an in vitro kinase assay as described in Materials and Methods . A recombinant active IKKβ isoform served as positive control. Shown are the mean values of absorption measured at a wavelength of 450 nm of three independent experiments −/+ SD. *, P<0.05. ( C ) HEK-293 cells were transfected with cDNAs coding for VSV-tagged Plexin-B1 (VSV-PlxnB1), FLAG-tagged PDZ-RhoGEF (FLAG-PRG) and NF-κB-dependent luciferase reporter plasmid (NF-κB-Luc). 48 hours after transfection, cells were incubated without (−) or with (+) TNFα (25 ng/ml) or Sema4D (150 nM) for 8 hours followed by the photometric quantification of reporter luciferase activity. ( D ) Wild-type MCF-7 cells (WT) and MCF-7 cells transduced with a degradation-resistent dominant-negative IκBα mutant (S32A/S36A) were serum-depleted, incubated in the absence (−) or presence (+) of 25 ng/ml TNFα or 150 nM Sema4D for 20 minutes and lysed. Lysates were probed with anti-IκBα antibody (left panel) to test the expression and functionality of the IκBα mutant or were immunoblotted with an anti-phospho-ErbB-2 antibody to visualize phosphorylated ErbB-2 and with an anti-ErbB-2 antibody to control expression levels (right panel). Protein levels were controlled by immunoblotting with an anti-α-tubulin antibody. ( E ) MCF-7 cells were preincubated with 25 µM of NF-κB inhibitor SN50 for the indicated time periods. Thereafter, cells were treated with control buffer (−) or 150 nM Sema4D (+) for 20 minutes, lysed and ErbB-2 phosphorylation was analyzed as described (left panel). To test the functionality of the NF-κB inhibitor, HEK-293 cells were transfected with a NF-κB dependent luciferase reporter plasmid (NF-κB-Luc) (right panel). After preincubation with 25 µM SN50 for 120 minutes, HEK-293 cells were incubated in the absence (−) or presence (+) of 25 ng/ml TNFα for 8 hours and luciferase acitivity was quantified. Shown are the mean values of three independent experiments −/+ SD. *, P<0.05.

Article Snippet: For precipitation of endogenous Plexin-B1 in MCF-7 cells, we used an anti-Plexin-B1 antibody (R&D Systems).

Techniques: Incubation, Control, In Vitro, Kinase Assay, Recombinant, Positive Control, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Transduction, Dominant Negative Mutation, Mutagenesis, Expressing, Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA

doi: 10.1371/journal.pone.0105661

Figure Lengend Snippet: ( A ) HEK-293 cells were transfected with cDNAs encoding VSV-Plexin-B1 (VSV-PlxnB1), FLAG-PDZ-RhoGEF (FLAG-PRG) alone or together with a HA-tagged kinase-deficient IKKα-mutant (HA-IKKα-KD). After incubation in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, VSV-Plexin-B1 was immunoprecipitated (IP) using an anti-VSV antibody and precipitates were immunoblotted (IB) using anti-ErbB-2, anti-VSV or anti-HA antibodies. Shown are the autoluminograms of immunoblots stained with the indicated antibodies. ( B ) MCF-7 cells were incubated with buffer (−) or IKK inhibitor SC-514 (50 µM) for 30 minutes. Thereafter, cells were stimulated without (−) or with 150 nM Sema4D (+) for 20 minutes, lysed, and endogenous Plexin-B1 was immunoprecipitated using an anti-Plexin-B1 antibody. Shown are Western blots of lysed or immunoprecipitated (IP) samples stained with the indicated antibodies (IB). ( C ) 48 hours after transfection with cDNAs encoding truncated versions of VSV-Plexin-B1 (VSV-PlxnB1ΔIC) and HA-ErbB-2 (HA-ErbB-2ΔIC), HEK293 cells were treated without (−) or with IKK inhibitor – SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes. Thereafter, cells were incubated in the absence (−) or presence (+) of 150 nM Sema4D for 20 minutes, lysed and immunoprecipitated (IP) using an anti-VSV antibody. Precipitates were seperated by SDS-PAGE and analyzed by immunoblotting (IB) with anti-VSV- or anti-HA- antibodies. ( D ) MDA-MB-468 cells were incubated without (−) or with SC-514 (50 µM) or NBDBP (100 µM) for 30 minutes, and Plexin-B1 was then immunoprecipitated (IP) from lysed cells. Precipitates were analysed by SDS-PAGE and immunoblotted with antibodies against c-Met or Plexin-B1. Shown are representative examples of at least three experiments.

Article Snippet: For precipitation of endogenous Plexin-B1 in MCF-7 cells, we used an anti-Plexin-B1 antibody (R&D Systems).

Techniques: Transfection, Mutagenesis, Incubation, Immunoprecipitation, Western Blot, Staining, SDS Page

Journal: PLoS ONE

Article Title: The IκB Kinase Complex Is Required for Plexin-B-Mediated Activation of RhoA

doi: 10.1371/journal.pone.0105661

Figure Lengend Snippet: ( A ) BT-474 and MT-2 cells were treated with control buffer, 25 µM SC-514 or 13 µM NBDBP for 30 minutes and lysed. Cell lysates were immunoblotted using anti-IκBα and anti-α-tubulin antibodies. ( B ) BT-474 and MT-2 cells were transfected with control or Plexin-B1 siRNA. 48 hours later, cells were starved for 12 hours and treated with IKK-inhibitors as described above. Cells were lysed, and the levels of activated RhoA were determined as described in the Materials and Methods . Shown are representative examples of at least three experiments. In a parallel experiment cells were lysed and Plexin-B1 expression in MT-2 cells was tested using RT-PCR. ( C, D ) Non-transfected BT-474 (C) and MT-2 (D) cells or cells transfected with control siRNA or siRNA directed against Plexin-B1 were counted and 1×10 5 (BT474) or 3×10 4 (MT-2) cells were plated in transwell invasion inserts. Non-transfected cells were treated without or with IKKβ/γ inhibitors as described. After 24 hours, invaded cells were fixed with 4% PFA, stained with Hoechst 33342 and counted. Data are expressed as mean values −/+ SD from triplicates. *, P<0.05

Article Snippet: For precipitation of endogenous Plexin-B1 in MCF-7 cells, we used an anti-Plexin-B1 antibody (R&D Systems).

Techniques: Control, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining