rabbit polyclonal anti-n-wasp antibodies  (Santa Cruz Biotechnology)

Journal: PLoS ONE

Article Title: Identification of Shigella flexneri IcsA Residues Affecting Interaction with N-WASP, and Evidence for IcsA-IcsA Co-Operative Interaction

doi: 10.1371/journal.pone.0055152

Figure Lengend Snippet: (A) Whole cell lysates from mid-exponential phase cultures of the indicated S. flexneri strains were subjected to Western immunoblotting with anti-IcsA antibody. S = S-LPS; R = R-LPS. IcsA − = IcsA deletion control. The 120 kDa band corresponds to the full length IcsA and the 85 kDa band corresponds to the cleaved form (IcsA’). (B) HeLa cells were infected with mid-exponential phase S. flexneri ΔicsA expressing the IcsA mutants and formalin fixed. HeLa cells and bacteria nuclei were labelled with DAPI (blue), F-actin was labelled with Alexa Fluor 488-phalloidin (green), and N-WASP was labelled with anti-N-WASP antibody and Alexa Fluor 594-conjugated donkey anti-rabbit antibody (red) as detailed in Materials and Methods. IF images were observed at 100×magnification. Arrows indicate N-WASP recruitment and F-actin comet tail formation. Insert shows an enlargement of the indicated region. Strains were assessed in two independent experiments. Scale bar = 10 µm. (C) Plaque assay by S. flexneri ΔicsA strains expressing IcsA::BIO, IcsA::BIO G331W or IcsA::BIO V382R. Confluent HeLa cell monolayers were infected with mid-exponential phase S. flexneri strains for 2 h, and plaques were observed 48 h post-infection as detailed in Materials and Methods. 30 plaques were measured from each experiment. Data are represented as mean ± SEM of three independent experiments. ***, P <0.001 (determined by Student’s unpaired one-tailed t test).

Article Snippet: The rabbit polyclonal anti-N-WASP and monoclonal anti-Arp3 (BD Biosciences) antibodies were both used at 1∶100 in IF.

Techniques: Western Blot, Infection, Expressing, Plaque Assay, One-tailed Test

Journal: PLoS ONE

Article Title: Identification of Shigella flexneri IcsA Residues Affecting Interaction with N-WASP, and Evidence for IcsA-IcsA Co-Operative Interaction

doi: 10.1371/journal.pone.0055152

Figure Lengend Snippet: (A) HeLa cells were infected with mid-exponential phase S. flexneri ΔicsA (S-LPS) or S. flexneri Δ icsA Δ rmlD (R-LPS) having an empty vector or expressing IcsA::BIO, IcsA::BIO Y716F, IcsA::BIO Y716G or IcsA::BIO D717G, and then formalin fixed. HeLa cells and bacteria nuclei were labelled with DAPI (blue), F-actin was labelled with Alexa Fluor 488-phalloidin (green), and N-WASP was labelled with anti-N-WASP antibody and Alexa Fluor 594-conjugated donkey anti-rabbit antibody (red) as detailed in Materials and Methods. IF images were observed at 100×magnification. Arrows indicate N-WASP recruitment and F-actin comet tail formation. Enlargements of relevant region shown for clarity. Strains were assessed in two independent experiments. Scale bar = 10 µm. (B) Plaque assay of S. flexneri ΔicsA strains expressing IcsA::BIO, IcsA::BIO Y716F, IcsA::BIO Y716G or IcsA::BIO D717G. Confluent HeLa cell monolayers were infected with mid-exponential phase S. flexneri strains for 2 h, and plaques were observed 48 h post-infection as detailed in Materials and Methods. 30 plaques were measured from each experiment. Data are represented as mean ± SEM of three independent experiments. ***, P <0.001 (determined by Student’s unpaired one-tailed t test).

Article Snippet: The rabbit polyclonal anti-N-WASP and monoclonal anti-Arp3 (BD Biosciences) antibodies were both used at 1∶100 in IF.

Techniques: Infection, Plasmid Preparation, Expressing, Plaque Assay, One-tailed Test

Journal: PLoS ONE

Article Title: Identification of Shigella flexneri IcsA Residues Affecting Interaction with N-WASP, and Evidence for IcsA-IcsA Co-Operative Interaction

doi: 10.1371/journal.pone.0055152

Figure Lengend Snippet: (A, B) IF microscopy to detect N-WASP, Arp3 recruitment and F-actin comet tail formation by intracellular S. flexneri strains. HeLa cells were infected with mid-exponential phase S. flexneri ΔicsA ΔrmlD (R-LPS) strains expressing either IcsA::BIO only or co-expressing IcsA::BIO V382R (N-WASP IR II) and IcsA::BIO Y716G D717G (N-WASP IR III), and formalin fixed. HeLa cells and bacteria nuclei were labelled with DAPI (blue), and N-WASP was labelled with anti-N-WASP antibody and either (A) Alex Fluor 488-conjugated donkey anti-rabbit antibody (green) or (B) Alex Fluor 594-conjugated donkey anti-rabbit antibody (red). (A) Arp3 was labelled with anti-Arp3 monoclonal antibody and an Alex Fluor 594-conjugated donkey anti-mouse antibody (red). (B) F-actin was labelled with Alexa Fluor-488-phalloidin (green). IF images were observed at 100×magnification. Arrows indicate N-WASP, Arp3 recruitment and F-actin tail formation. Enlargements of relevant region shown for clarity. Strains were assessed in two independent experiments. Scale bar = 10 µm. (C) Quantification of N-WASP/Arp3 recruitment, and (D) N-WASP/F-actin tail or capping formation, by intracellular S. flexneri ΔicsA ΔrmlD strains expressing IcsA::BIO only or co-expressing IcsA::BIO V382R (N-WASP IR II) and IcsA::BIO Y716G D717G (N-WASP IR III). Bacteria that either recruited both N-WASP and Arp3 (C), or recruited N-WASP and formed F-actin comet tail/capping (D), were scored from infected HeLa cells ( n = 20 HeLa cells; ∼250–350 bacteria). Data are represented as percentage of N-WASP/Arp3 recruitment ± SEM of two independent experiments (C); and, as percentage of N-WASP recruitment/F-actin tail or capping formation ± SEM of two independent experiments (D). **, 0.001< P <0.01; ***, P <0.001 (determined by Student’s unpaired one-tailed t test). IR II & III = Interacting region II and III ( S. flexneri co-expressing IcsA::BIO V382R and IcsA::BIO Y716G D717G).

Article Snippet: The rabbit polyclonal anti-N-WASP and monoclonal anti-Arp3 (BD Biosciences) antibodies were both used at 1∶100 in IF.

Techniques: Microscopy, Infection, Expressing, One-tailed Test

Journal: Neuron

Article Title: Parkinson Sac Domain Mutation in Synaptojanin 1 Impairs Clathrin Uncoating at Synapses and Triggers Dystrophic Changes in Dopaminergic Axons

doi: 10.1016/j.neuron.2017.01.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: rabbit polyclonal anti-nWASP , ECM biosciences , Cat# WP2101; RRID:AB_715260.

Techniques: Recombinant, Sequencing, Software