polyclonal rabbit anti-human epo antibody  (Accurate Chemical & Scientific Corporation)

Journal: bioRxiv

Article Title: Structural and functional characterization of the NF-κB-targeting toxin AIP56 from Photobacterium damselae subsp. piscicida reveals a novel mechanism for membrane interaction and translocation

doi: 10.1101/2023.05.04.539443

Figure Lengend Snippet: (A) Localization of the putative pH sensing residues selected for replacement on the three-dimensional structure of the AIP56 catalytic domain. Cartoon (left) and surface representation of AIP56 catalytic domain with (middle) or without (right) the middle domain. The catalytic residues (H165, E166 and H169) are shown in red, the putative pH sensing residues in orange and the D209-K247 hairpin in marine blue. The cysteine residues (pink sticks) forming the disulfide bridge (yellow) are also shown. (B) Analysis of NF-kB p65 cleavage in mouse bone marrow-derived macrophages (mBMDM) by V5 plus His-tagged AIP56 variants. Cleavage of p65 was assessed by western blotting (upper panel; chromogenic detection) and protein loading by staining the membranes with Ponceau S (lower panel). The result shown is representative of six independent experiments. (C) Peak ANS fluorescence measured at 475 nm for the indicated pH, normalized by subtracting the corresponding values at pH 7 (fluorescence due to conformational changes caused by the mutation and not due to acidification). The measurement curves for each pH at different wavelengths are shown in Fig. S7A. The results shown are representative of at least three independent experiments. (D) Coomassie Blue-stained SDS-PAGE gels from limited proteolysis of AIP56 and AIP56 H231K/E234K by Proteinase K. A and B mark the bands corresponding to the catalytic and receptor-binding domains, respectively. (E) Interaction of AIP56 or AIP56 variants with black lipid bilayers. Single channel recordings of DiPhPC/n-decane membranes after addition of the indicated proteins to one side of the black lipid bilayer at a final concentration of 14 nM. Membrane activity was induced by acidification (pH 4.8; red arrows) of the aqueous phase at the cis-side of the chamber. Each result shown is representative of at least three independent measurements.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (c-20) rabbit polyclonal antibody (sc-372) was from Santa Cruz Biotechnology and the anti-V5 (R960-25) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Derivative Assay, Western Blot, Staining, Fluorescence, Mutagenesis, SDS Page, Binding Assay, Concentration Assay, Membrane, Activity Assay

Journal: bioRxiv

Article Title: Structural and functional characterization of the NF-κB-targeting toxin AIP56 from Photobacterium damselae subsp. piscicida reveals a novel mechanism for membrane interaction and translocation

doi: 10.1101/2023.05.04.539443

Figure Lengend Snippet: (A) Schematic representation of chimera Bla L19-W286 AIP56 P210-N497 . (B) FRET-based assay to access the effect of Hsp90 inhibition on Bla delivery. The cleaved/uncleaved CCF4-AM ratios were determined by quantifying a minimum of 10 microscopic fields per condition. Results shown represent one out of three independent experiments. Statistical significance was tested by Kruskal-Wallis nonparametric test and the adjusted p values for individual comparisons were obtained by Bonferroni correction. P values are indicated; ns = non-significant. (C) Control of 17-DMAG activity by confirming its inhibitory effect on NF-kB p65 cleavage upon AIP56 intoxication of mBMDM. A representative blot of three independent experiments is shown. Loading correction was achieved by dividing the density of p65 by the respective density of the Ponceau S staining. Values are mean ± SD.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (c-20) rabbit polyclonal antibody (sc-372) was from Santa Cruz Biotechnology and the anti-V5 (R960-25) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Inhibition, Control, Activity Assay, Staining

Journal: bioRxiv

Article Title: Structural and functional characterization of the NF-κB-targeting toxin AIP56 from Photobacterium damselae subsp. piscicida reveals a novel mechanism for membrane interaction and translocation

doi: 10.1101/2023.05.04.539443

Figure Lengend Snippet: (A) V5 plus His-tagged AIP56 modified in the aspartate-rich motif (AIP56 D274S/D276-278S and AIP56 D274N/D276-278N ) is unable to cleave p65 in intact cells. Cleavage of p65 was assessed by western blotting and protein loading by Ponceau S staining. The result shown is representative of six independent experiments. (B) V5 plus His-tagged AIP56 D274S/D276-278S and AIP56 D274N/D276-278N were unable to translocate across the host cell membrane in response to acidification. In all experiments, mock-treated cells were used as controls. NF-kB p65 cleavage was analyzed by western blotting. The result shown is representative of five independent experiments. The box plot shows the quantification of intact NF-kB p65 normalized for Ponceau S. Statistical significance was tested by one-way ANOVA and p values for the individual comparisons were calculated using Tukey’s HSD test. P values are indicated, ns = non-significant. (C) AIP56 D274S/D276-278S and AIP56 D274N/D276-278N retained the ability to interact with black lipid bilayers. Proteins were used at a final concentration of 14 nM. Membrane activity was induced by acidification (pH 4.8; red arrows) of the aqueous phase at the cis-side of the chamber. Each result shown is representative of at least three independent measurements.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (c-20) rabbit polyclonal antibody (sc-372) was from Santa Cruz Biotechnology and the anti-V5 (R960-25) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Modification, Western Blot, Staining, Membrane, Concentration Assay, Activity Assay