Journal: Methods in Molecular Biology

Article Title: Semaphorin Signaling

doi: 10.1007/978-1-4939-6448-2

Figure Lengend Snippet: Fig. 1 Gel fi ltration data for intracellular plexin- B1 [ 17 ]. ( a ) and inset : Calibration. The gel fi ltration column was calibrated using fi ve standard molecular weight proteins and a relationship between elution volumes (partition coeffi cient Kav) and molecular weight (Mr) is established. ( b ) Data for the unbound intracellular region of plexin-B1, Rac1 .Q61L, and their association. Plexin-B1 is prone to form higher order oligomers in the presence of the GTPase (peak of complex at approx. 1.25 mL). This is consistent with the suggestion of Jones and coworkers that Rac1 stabilizes plexin-B1 in a trimeric complex, although they were unable to observe such a complex in solution [ 13 ]. A version of this fi gure was originally published in J. Biol.Chem. Tong, Y et al., Structure and function of the intracellular region of the plexin-B1 transmembrane receptor. 284: 35962-35972. © The American Society for Biochemistry and Molecular Biology

Article Snippet: To immunoprecipitate Plexin-D1, use 1 μg of Plexin- D1 antibody (goat polyclonal, R&D Systems, AF4160).

Techniques: Molecular Weight

Journal: Methods in Molecular Biology

Article Title: Semaphorin Signaling

doi: 10.1007/978-1-4939-6448-2

Figure Lengend Snippet: Fig. 2 Interaction of Rac1. Q61L with the plexin-B1 intracellular region ( a , b ) and the plexin-B1 RBD ( c , d ). Equilibrium binding between Rac1. Q61L and plexin-B1 intracellular region or PB1-RBD was characterized using SPR. The plexin was immobilized on a SA chip using the capturing approach of biotinylated ligands on a streptavidin surface with an immobilization density of 406 RU for the plexin-B1 intracellular region and 181 RU for PB1-RBD. 0.25–128 μM of Rac1 were fl own over the surface at 25 °C. The dissociation constant was calculated to be 23.2 (±0.23) μM for the PB1 intracellular region and 15.1 (±0.15) μM for PB1- RBD

Article Snippet: To immunoprecipitate Plexin-D1, use 1 μg of Plexin- D1 antibody (goat polyclonal, R&D Systems, AF4160).

Techniques: Binding Assay

Journal: Methods in Molecular Biology

Article Title: Semaphorin Signaling

doi: 10.1007/978-1-4939-6448-2

Figure Lengend Snippet: Fig. 3 Isothermal titration calorimetry measurements of the binding affi nity between the interaction of the GTPase Rac1 .Q61L constitutive active mutant and two plexin constructs [ 11 , 17 ]. Binding isotherm for Rac1 association with PB1- RBD ) ( a ) and with plexin- B1 intracellular region ( b ). Shown are representative raw data ( top ) as well as the peak integrated and concentration-normalized enthalpy change ( bottom ). Similarly to the SPR data, the RBD binds the GTPase more tightly than the intracellular region (Kd 6.1 μM vs. 43 μM), suggest- ing that the binding site is partly occluded in the intracellular protein. A version of this fi gure was originally published in J Biol Chem panel ( a ): Tong, Y et al., Binding of Rac1, Rnd1 , and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-B1 effector domain. 283: 37215–37224 and panel ( b ), Tong, Y et al., Structure and function of the intracellular region of the plexin-b1 transmembrane receptor . 284: 35962–35972. © The American Society for Biochemistry and Molecular Biology

Article Snippet: To immunoprecipitate Plexin-D1, use 1 μg of Plexin- D1 antibody (goat polyclonal, R&D Systems, AF4160).

Techniques: Isothermal Titration Calorimetry, Binding Assay, Mutagenesis, Construct, Concentration Assay

Journal: Methods in Molecular Biology

Article Title: Semaphorin Signaling

doi: 10.1007/978-1-4939-6448-2

Figure Lengend Snippet: Fig. 2 Single- and multiple-turnover plexin GAP activity reactions. ( a ) Example data set from a single-turnover GAP activity measurement using 10 μM mouse PlexinC1 cyto and 70 μM human Rap1B. Measurement of Rap1B with no plexin is used to establish the basal GTP hydrolysis rate. The GAP reaction proceeds as a single expo- nential and reaches completion at around 200 s. ( b ) Data from a multiple-turnover GAP activity assay using 15 μM of a human Rap1B/zebrafi sh PlexinC1 cyto fusion protein. Rap1B basal hydrolysis is measured as in ( a )

Article Snippet: To immunoprecipitate Plexin-D1, use 1 μg of Plexin- D1 antibody (goat polyclonal, R&D Systems, AF4160).

Techniques: Activity Assay

Journal: Methods in Molecular Biology

Article Title: Semaphorin Signaling

doi: 10.1007/978-1-4939-6448-2

Figure Lengend Snippet: Fig. 2 Examples of immunoblots following immunoprecipitation of Plexin- B1 and RhoA pulldown . MCF-7 cells were treated without (−) or with Sema4D (+). After incubation for 20 min, cells were lysed. ( a ) Plexin-B1 was immunoprecipitated using a Plexin-B1 antibody (IP), and precipitates were immunoblotted (IB) using Plexin-B1 and phospho-tyrosine (pY) antibodies. In addition, lysates were immu- noblotted using a Plexin-B1 antibody. ( b ) After lysis, active Rho was precipitated using GST- RBD (pulldown). The pulldowns and lysates were immunoblotted (IB) using a RhoA antibody

Article Snippet: To immunoprecipitate Plexin-D1, use 1 μg of Plexin- D1 antibody (goat polyclonal, R&D Systems, AF4160).

Techniques: Western Blot, Immunoprecipitation, Incubation, Lysis

Journal: Methods in Molecular Biology

Article Title: Semaphorin Signaling

doi: 10.1007/978-1-4939-6448-2

Figure Lengend Snippet: Fig. 1 COS7 cells expressing membrane-targeted EGFP and Plexin- D1 . ( a ) Non- collapsed cells without stimulation (i.e., without addition of Sema3E conditioned media). ( b ) Collapsed cells after 30-min treatment with Sema3E conditioned media

Article Snippet: To immunoprecipitate Plexin-D1, use 1 μg of Plexin- D1 antibody (goat polyclonal, R&D Systems, AF4160).

Techniques: Expressing, Membrane

Journal: bioRxiv

Article Title: Endothelial ADAM17 Promotes Neutrophil Migration and Pulmonary Microvascular Permeability in ARDS

doi: 10.64898/2026.01.21.700786

Figure Lengend Snippet: Flow cytometric analysis of PMN distribution across lung compartments—blood (A) , endothelial-adherent (B) , interstitial (C) , and BAL (D) -24 hours after LPS inhalation with or without systemic ADAM17 inhibition (n = 4/13–14/7/7). (E) Quantification of CXCL2/3 in BAL fluid three hours after LPS inhalation by ELISA (n = 4/12/7/7). (F) Quantification of JAM-A MFI on pulmonary endothelial cells 24 hours after LPS inhalation (n = 4/14/7/7). (G) Quantification of VE-cadherin MFI in lung tissue using LasX (n = 4 mice per group; 10 measurements per image). (H–I) Photometric quantification of Evans blue in plasma (H) and lung tissue (I) six hours after LPS inhalation (n = 4/11/7/6–7). The data shown for ADAM17 fl/fl Tie2-Cre - controls were previously presented in the following panels: in , in , in , and in (to minimize animal numbers in accordance with the 3R principle). Data are presented as mean ± SD; Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, using one-way ANOVA.

Article Snippet: Sections were then incubated with primary antibodies targeting ADAM17 (rabbit anti-ADAM17, polyclonal, Bioss, USA), Ly6G/C (rat anti-Ly6G/C, clone RB6-8C5, Abcam, UK), VE-Cadherin (goat anti-VE-Cadherin, polyclonal, R&D Systems, USA), TNFR1 (mouse anti-TNFR1, clone H-5, Santa Cruz, USA), IL-6Rα (mouse anti-IL-6Rα, clone H-7, Santa Cruz, USA), and vWF (sheep anti-vWF, polyclonal, Abcam, UK) followed by incubation with the corresponding fluorescently labelled secondary antibodies including Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 647 goat anti-rat, Alexa Fluor 488 donkey anti-goat, Alexa Fluor 594 goat anti-mouse, Alexa Fluor 488 donkey anti-mouse and Alexa Fluor 647 donkey anti-sheep.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Clinical Proteomics