anti cnf1 antibody  (Hycult Biotech)

Journal: Gut microbes

Article Title: The cnf1 gene is associated with an expanding Escherichia coli ST131 H 30Rx/C2 subclade and confers a competitive advantage for gut colonization.

doi: 10.1080/19490976.2022.2121577

Figure Lengend Snippet: Figure 1. Prevalence overtime in representative E. coli sequence types bearing cnf1. Bar chart show number of E. coli strains from ST131, ST73, ST12 and ST127 isolated each year during the period 2002–2019, left y-axis. Percentages of cnf1-positive strains per year, right y-axis.

Article Snippet: Membranes were incubated with the primary antibody: CNF1 (Santa Cruz sc52655 clone NG8 1/1000), RNA Polymerase (Biolegend 699907 clone NT73 1/ 1000) and rabbit serum (1/1000) against the conserved amino acids 914–936 of HlyA, as previously described.79 Membranes were washed with TBS-T and incubated with horseradish peroxidase (HRP)- conjugated secondary antibodies for 1 h. Signals were observed using Immobion Western Chemiluminescent HRP Substrate (Merck).

Techniques: Sequencing, Isolation

Journal: Gut microbes

Article Title: The cnf1 gene is associated with an expanding Escherichia coli ST131 H 30Rx/C2 subclade and confers a competitive advantage for gut colonization.

doi: 10.1080/19490976.2022.2121577

Figure Lengend Snippet: Figure 2. Dynamic of CNF1-encoding gene in E. coli ST131 from EnteroBase. A) Maximum likelihood phylogeny of E. coli ST131 from EnteroBase (Sup. Figure S2 for extended information). The phylogeny was constructed with 5,231 genomes for a total of 37,304 non-recombinant core-genome SNPs. The different clades and subclades A, B, C0, C1, C2_0, C2_1, C2_2 are highlighted in blue, red, light green, green, pink, Orange and purple respectively. From inside to outside circles are indicated (1) fimH alleles, (2) gyrA and parC alleles conferring resistance to fluoroquinolones (shown in green), (3) positive strains for blaCTX-M-15 (shown in Orange) and (4) strains bearing cnf1 gene (shown in black).) Hierarchical clustering of strains from clade C (n = 3981 strains) based on their accessory gene content. The pan-genome is composed of 51,742 genes including 2,672 genes that are present in 98% of the strains. The graph displays the 7,678 genes identified as present in at least 50 and less than 3,930 genomes. The colored annotation indicates (from left to right) the presence of cnf1 (CNF1_status), subclades (C1, C1 CNF1_LL1, C2_0, C2_1, C2_1 CNF1_LL2, C2_2) and accessory genes cluster (AG_clusters). Large lineages of cnf1-positive strains in clades C1 and C2_1 are denoted CNF1_LL1 and CNF1_LL2, respectively. Red stars indicate the two large lineages of cnf1-positive strains.

Article Snippet: Membranes were incubated with the primary antibody: CNF1 (Santa Cruz sc52655 clone NG8 1/1000), RNA Polymerase (Biolegend 699907 clone NT73 1/ 1000) and rabbit serum (1/1000) against the conserved amino acids 914–936 of HlyA, as previously described.79 Membranes were washed with TBS-T and incubated with horseradish peroxidase (HRP)- conjugated secondary antibodies for 1 h. Signals were observed using Immobion Western Chemiluminescent HRP Substrate (Merck).

Techniques: Construct, Recombinant

Journal: Gut microbes

Article Title: The cnf1 gene is associated with an expanding Escherichia coli ST131 H 30Rx/C2 subclade and confers a competitive advantage for gut colonization.

doi: 10.1080/19490976.2022.2121577

Figure Lengend Snippet: Figure 4. Increase over the years in the proportion of cnf1-positive strains in E. coli ST131 H30Rx/C2. A) Distribution of fimH alleles (upper panel) or clades/subclades (lower panel) within the study population of E. coli ST131. Both figures show observed counts per year (dots) and data fitted lines (dashed lines) with a generalized linear model (Poisson regression). B) Increase of the proportion of cnf1-positive strains in the whole E. coli ST131 population along time (top panel, P = 7.41 10–7) and by clades and subclades. The black dots represent the observed proportion of cnf1-positive strains by year with fitted line of a logistic regression model (blue curves). Dashed gray lines display the 95% confidence intervals. The P-values are not significant for clade A (P = 0.287), B (P = 0.952), H30R/C1 (P = 0.992) and significant for H30Rx/C2 (P = 1.25 10−11).

Article Snippet: Membranes were incubated with the primary antibody: CNF1 (Santa Cruz sc52655 clone NG8 1/1000), RNA Polymerase (Biolegend 699907 clone NT73 1/ 1000) and rabbit serum (1/1000) against the conserved amino acids 914–936 of HlyA, as previously described.79 Membranes were washed with TBS-T and incubated with horseradish peroxidase (HRP)- conjugated secondary antibodies for 1 h. Signals were observed using Immobion Western Chemiluminescent HRP Substrate (Merck).

Techniques:

Journal: Toxins

Article Title: CTX-CNF1 Recombinant Protein Selectively Targets Glioma Cells In Vivo

doi: 10.3390/toxins13030194

Figure Lengend Snippet: CNF1 maintains its activity on glioma cells even when inserted in the CTX-CNF1 recombinant molecule. ( A ) Structure of CTX-CNF1. Chlorotoxin (CTX) is located at the N-terminus of the recombinant molecule and is followed by CNF1, formed by its three domains (binding, translocation and catalytic). ( B ) Percentage of multi- and mononucleated GL261 cells in control (grey, PBS) and after 48 h of CTX-CNF1 administration (green, 25 nM). Inset, representative images of the two conditions, scale bar = 4μm. ( C ) Percentage of vital GL261 cells in control (grey, PBS) and in 48 h treatment of CTX-CNF1 (green) at different concentrations (i.e., 1, 12, 25, 30, 40 and 50 nM). Data represent means ± SEM, one way ANOVA p < 0.001. ( D ) Percentage of betagalactosidase positive GL261 cells in control (grey, PBS) and after CTX-CNF1 administration (green, 25 nM) at different time points (i.e., 24 h, 48 h, 72 h, 6 d). Data represent means ± SEM, One Way ANOVA p < 0.001. ( E ) Quantitative RT-PCRs showing the relative expression of the senescence markers p21 (Cdkn1a, cyclin-dependent kinase inhibitor (1) and p16 (Cdkn2a, cyclin-dependent kinase inhibitor 2) in GL261 cells (vehicle, grey; CTX-CNF1, green). Data represent means ± SEM, t test (p16, p = 0.0001; p21, p = 0.0164). ( F ) Increased expression of p16 (Cdkn2a, cyclin-dependent kinase inhibitor 2) in U87 cells treated with CTX-CNF1 (green) with respect to vehicle (grey). Data represent means ± SEM, t test ( p = 0.013). Total cells ( G ) and spheroids ( H ) number of PDGF+ TRP53−/− cells in control (grey, PBS) and after 25 nM of CTX-CNF1 (green) at 72 h and 6 d. Data represent means ± SEM, One Way ANOVA p < 0.001. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Briefly, protein extracts were separated by electrophoresis and blotted; filters were blocked and incubated overnight at 4 °C with anti-CNF1 catalytic domain primary antibodies (1:1000, ThermoFisher, Waltham, MA, USA) and probed with anti-tubulin antibody (1:40,000, Cell Signaling, Danvers, MA, USA) as an internal standard for protein quantification.

Techniques: Activity Assay, Recombinant, Binding Assay, Translocation Assay, Control, Expressing

Journal: Toxins

Article Title: CTX-CNF1 Recombinant Protein Selectively Targets Glioma Cells In Vivo

doi: 10.3390/toxins13030194

Figure Lengend Snippet: Increased survival of CTX-CNF1 glioma-bearing mice. ( A , C , E , G ) Experimental protocols. GL261 cells were injected in visual cortex and, 12 days after glioma induction, different treatments were administered. ( A , C ) Glioma-bearing mice were treated intraventricularly ( A , B ) or intravenously ( C , D ) with CTX-CNF1 (80 nM) or vehicle solution (PBS). ( B , D ) Survival rate of untreated and treated glioma-bearing mice. Note the increased surviving percentage of both CTX-CNF1 treated glioma-bearing mice ( B , p = 0.0018; D , p = 0.013). * p < 0.05; ** p < 0.01. ( F , H ) Survival rate of untreated and intravenously treated ( F, with CNF1; H , with CTX) glioma-bearing animals. We did not find any significant increase in survival after CNF1 ( F , p = 0.1); conversely, CTX treatment was able to increase survival rate of glioma-bearing animals ( H , p = 0.03). ( I ) Normalized fraction of tumor area occupied by KI67+ cells in control and in animals intravenously treated with CTX-CNF1 or CTX alone, 24 h and 48 h after the toxin administration (one way ANOVA p < 0.001). Data represent mean ± SEM. *** p < 0.001. ( L , M ) Number of peritumoral Iba1+ ( L , p = 0.19) and Mac2+ ( M , p < 0.001) cells in mice injected with GL261 cells into the visual cortex and systemically treated with CTX-CNF1 with respect to controls. Data represent mean ± SEM. *** p < 0.001.

Article Snippet: Briefly, protein extracts were separated by electrophoresis and blotted; filters were blocked and incubated overnight at 4 °C with anti-CNF1 catalytic domain primary antibodies (1:1000, ThermoFisher, Waltham, MA, USA) and probed with anti-tubulin antibody (1:40,000, Cell Signaling, Danvers, MA, USA) as an internal standard for protein quantification.

Techniques: Injection, Control

Journal: Toxins

Article Title: CTX-CNF1 Recombinant Protein Selectively Targets Glioma Cells In Vivo

doi: 10.3390/toxins13030194

Figure Lengend Snippet: CTX-CNF1 specificity of action on glioma cells. Representative image ( A ) and magnification ( B ) of a triple staining for chimeric protein (red), glioma PDGF+ TRP53 −/− GFP + cells (green) and cellular nuclei (blue). Note the high selectivity of CTX-CNF1 for recognizing and entering into GB mass. CTX-CNF1 was intravenously delivered and the pictures were taken 24 h after chimeric administration. ( A ) Scale bar = 1000 μm. ( B ) Scale bar = 50 μm. ( C ) In vivo image taken of PDGF + TRP53 −/− GFP + cells 24 h after CTX-CNF1 intravenous delivery. Scale bar = 5 μm ( D ) In vivo image taken of GL261 cells 6 days after CTX-CNF1 intravenous delivery. Scale bar = 10 μm. ( E ) Representative western blot: 12 days after GL261 injection, CTX-CNF1 administration were performed (left, intraventricular; right, intravenous). 48 h after the treatment, the chimeric protein is clearly detectable only in the tumoral mass, even when its administration was made through the caudal vein. Tum = tumor; PTum = peritumoral; Mot = motor area; CD = catalytic domain; GL261 = GL261 cells lysate untreated.

Article Snippet: Briefly, protein extracts were separated by electrophoresis and blotted; filters were blocked and incubated overnight at 4 °C with anti-CNF1 catalytic domain primary antibodies (1:1000, ThermoFisher, Waltham, MA, USA) and probed with anti-tubulin antibody (1:40,000, Cell Signaling, Danvers, MA, USA) as an internal standard for protein quantification.

Techniques: Staining, In Vivo, Western Blot, Injection

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: Design, expression, and solubility of cytotoxic necrotizing factor 1 (CNF1) variants. ( A ) Scheme of engineered CNF1 constructs presenting the following N-terminal and C-terminal tags: Angiopep-2 (An2), spaced by the GGSSRSS linker; 8xHis fused to a Tobacco Etch Virus recognition site (H8). ( B ) Recombinant protein expression levels in E. coli BL21 (DE3). Total cellular extracts obtained at 15 °C after 18 h induction with 0.5 mM IPTG were separated by 10% SDS-PAGE and analyzed either by Coomassie staining ( B1 ) or western blot ( B2 ) using a monoclonal anti-CNF1 antibody (NG8). Induced cells expressing CNF1-H8; induced cells expressing An2-CNF1-H8; not induced recombinant cells harboring pET40b- cnf1-h8 . ( C ) Evaluation of CNF1 solubility in E. coli extracts. After cellular lysis, insoluble (P) and soluble (S) fractions were analyzed. The first two panels are relative to the solubility assay concerning CNF1-H8 expression carried out using SDS-PAGE ( C1 and C3 ) and western blot ( C2 and C4 ). The third and fourth images represent the SDS-PAGE and western blot relative to An2-CNF1-H8 solubility, respectively.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques: Expressing, Solubility, Construct, Virus, Recombinant, SDS Page, Staining, Western Blot, Lysis

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: CNF1 variants activity. ( A ) SDS-polyacrylamide gel ( A1 ) and western blot ( A2 ) of CNF1 variants. ( B ) Phase-contrast micrographs of HEp-2 cells treated with CNF1 variants, showing the multinucleating/ruffling effects. Scale bar: 10 μm.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques: Activity Assay, Western Blot

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: Entry of CNF1 variants into HEp-2 cells. ( A ) Graph showing the inhibition of variants entry by mCNF1. ( B ) Western blot analysis of HEp-2 cells treated with An2-CNF1-H8 and its controls (CNF1-H8 and wt CNF1) and stained with an antibody against CNF1, normalized as a function of GAPDH. Note that all the toxins are found as full length and 55 kDa C-ter portions after 24 h treatment ( B1 ). Over a longer period of time, that is 48 h of treatment, washing to eliminate any remaining molecules in the culture medium, and further culture for 24 h, the full-length portions completely disappeared while the 55 kDa C-ter portions were present at a much lower concentration ( B2 ). All in all, these results point at the fact that An2-CNF1-H8 follows the same route of entry of wt CNF1. Variants and wt CNF1 were used at 10 −10 M. Data on the graph represent the mean ± SEM from at least three independent experiments. *** p < 0.001.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques: Inhibition, Western Blot, Staining, Concentration Assay

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: An2-CNF1-H8 activity on endothelial cells. Fluorescence micrographs of HBEC-5i cells treated with the An2-CNF1-H8 variant and its controls. Note the same actin architecture in cells exposed to the wt CNF1 and to the variants, indicating the same activity. Scale bar: 10 μm.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques: Activity Assay, Fluorescence, Variant Assay

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: An2-CNF1-H8 impairs growth in U87MG GBM cells. ( A and B ) Graphs showing cell growth impairment by An2-CNF1-H8 ( A ) and CNF1 ( B ) on U87MG GBM cells. Note that, whereas controls cells increase in cell number in the function of time, cells treated with An2-CNF1-H8 and CNF1 stopped growing. Data on the graph represent the mean ± SEM from at least three independent experiments. *** p < 0.001.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques:

Journal: Toxins

Article Title: Cnf1 Variants Endowed with the Ability to Cross the Blood–Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma

doi: 10.3390/toxins12050291

Figure Lengend Snippet: Mitochondrial impact and toxicity of An2-CNF1-H8 on U87MG cells. ( A ) Fluorescence micrographs of U87MG cells treated with An2-CNF1-H8 and stained with phalloidin, to detect F-actin, Mitotracker, to detect mitochondria and Hoechst 33342 to stain nuclei. Note that the variant impacts on mitochondria that appear slightly fragmented and scattered throughout the cell body with respect to controls. ( B and C ) Western blot of U87MG cells treated with An2-CNF1-H8 and graph analyses, showing that ( B1 and B2 ) Tom20 was transiently increased, whereas OPA-1 protein was not modified by the An2-CNF1-H8; ( C1 and C2 ) the pro-apoptotic Bax protein, involved in mitochondrial outer membrane permeabilization, was increased whereas the senescence marker beta-gal remained unvaried. Proteins were normalized as a function of GAPDH. ** p < 0.01. Scale bar: 10 μm.

Article Snippet: For western blot analysis, an anti-CNF1 antibody (NG8, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000) was used following the manufacturer’s instructions.

Techniques: Fluorescence, Staining, Variant Assay, Western Blot, Modification, Membrane, Marker