Human Plexin B2 Antibody

Detection of Human Plexin B2 by Western Blot. Western blot shows lysates of human brain tissue. PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human Plexin B2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5329) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for Plexin B2 at approximately 170 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.

Plexin B2 in Human Colon Cancer Tissue. Plexin B2 was detected in immersion fixed paraffin-embedded sections of human colon cancer using 3 µg/mL Sheep Anti-Human Plexin B2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5329) overnight at 4 °C. Before incubation with the primary antibody tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained with the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS019) and counterstained with hematoxylin (blue). Specific labeling was localized to the plasma membrane and cytoplasm of epithelial cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Plexin B2 in Mouse Brain. Plexin B2 was detected in perfusion fixed frozen sections of mouse brain (choroid plexus) using 10 µg/mL Sheep Anti-Human Plexin B2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5329) overnight at 4 °C. Tissue was stained with the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). View our protocol for Fluorescent IHC Staining of Frozen Tissue Sections.

Western Blot Shows Human Plexin B2 Specificity by Using Knockout Cell Line. Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and Plexin B2 knockout HeLa cell line (KO). PVDF membrane was probed with 0.5 µg/mL of Sheep Anti-Human Plexin B2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5329) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for Plexin B2 at approximately 170 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. GAPDH (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human Plexin B2 by Knockdown Validated 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 (1×105 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. Image collected and cropped by CiteAb from the following publication (https://journals.biologists.com/jcs/article/doi/10.1242/jcs.192211/265234/Rnd3-induced-cell-rounding-requires-interaction), licensed under a CC-BY license. Not internally tested by R&D Systems.